Identifying new therapeutic agents

ABSTRACT

Disclosed herein are methods for identifying novel drug candidates.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional applicationSer. No. 61/427,626, filed Dec. 28, 2010, the entire contents of whichare hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The vast majority of small molecule drugs act by binding a functionallyimportant site on a target protein, thereby modulating the activity ofthat protein. For example, the cholesterol-lowering drugs statins bindthe enzyme active site of HMG-CoA reductase, thus preventing the enzymefrom engaging with its substrates The fact that many such drug/targetinteracting pairs are known may have misled some into believing that asmall molecule modulator could be discovered for most, if not all,proteins provided a reasonable amount of time, effort, and resources.This is far from the case. Current estimates hold that only about 10% ofall human proteins are targetable by small molecules. The other 90% arecurrently considered refractory or intractable toward small moleculedrug discovery. Such targets are commonly referred to as “undruggable.”Wolfson, Chemistry & Biology 16, 2009, 910-12. These undruggable targetsinclude a vast and largely untapped reservoir of medically importanthuman proteins. Thus, there exists a great deal of interest indiscovering new molecular modalities capable of modulating the functionof such undruggable targets.

Small molecules are limited in their targeting ability because theirinteractions with the target are driven by adhesive forces, the strengthof which is roughly proportional to contact surface area. Because oftheir small size, the only way for a small molecule to build up enoughintermolecular contact surface area to effectively interact with atarget protein is to be literally engulfed by that protein. Indeed, alarge body of both experimental and computational data supports the viewthat only those proteins having a hydrophobic “pocket” on their surfaceare capable of binding small molecules. In those cases, binding isenabled by engulfment. Not a single example exists of a small moleculebinding with high-affinity to a protein outside of a hydrophobic pocket.

Nature has evolved a completely unique strategy that allows a smallmolecule to interact with target proteins at sites other thanhydrophobic pockets. This strategy, typified by the naturally occurringimmunosuppressive drugs cyclosporine A, rapamycin, and FK506, initiallyinvolves the formation of a high-affinity complex of the small moleculewith a small presenting protein. The composite surface of the smallmolecule and the presenting protein then engages the target. Thus, forexample, the binary complex formed between cyclosporin A and cyclophilinA targets calcineurin with high affinity and specificity, but neithercyclosporin A or cyclophilin A alone binds calcineurin with measurableaffinity.

Many important therapeutic targets exert their function by complexationwith other proteins. The protein/protein interaction surfaces in many ofthese systems contain an inner core of hydrophobic side chainssurrounded by a wide ring of polar residues. The hydrophobic residuescontribute nearly all of the energetically favorable contacts, and hencethis cluster has been designated as a “hotspot” for engagement inprotein-protein interactions. Importantly, in the aforementionedcomplexes of naturally occurring small molecules with small presentingproteins, the small molecule provides a cluster of hydrophobicfunctionality akin to a hotspot, and the protein provides the ring ofmostly polar residues. In other words, presented small molecule systemsmimic the surface architecture employed widely in naturalprotein/protein interaction systems.

Nature has demonstrated the ability to reprogram the target specificityof presented small molecules—portable hotspots—through evolutionarydiversification. In the best characterized example, the complex formedbetween FK506 binding protein (FKBP) and FK506 targets calcineurin.However, FKBP can also form a complex with the related moleculerapamycin, and that complex interacts with a completely differenttarget, TorC1. To date, no methodology has been developed to reprogramthe binding and modulating ability of presenter protein/ligandinterfaces so that they can interact with and modulate other targetproteins that have previously been considered undruggable.

In addition, it is well established that some drug candidates failbecause they modulate the activity of both the intended target and othernon-intended proteins as well. The problem is particularly daunting whenthe drug binding site of the target protein is similar to binding sitesin non-target proteins. The insulin like growth factor receptor(IGF-1R), whose ATP binding pocket is structurally similar to thebinding pocket of the non-target insulin receptor (IR), is one suchexample. Small molecule development candidates that were designed totarget IGF-1R typically have the unacceptable side effect of alsomodulating the insulin receptor. However, structural dissimilarities doexist between these two proteins in the regions surrounding the ATPbinding pocket. Despite such knowledge, no methodology exists to date totake advantage of those differences and develop drugs that are specificto IGF-1R over IR.

SUMMARY OF THE INVENTION

The present invention provides, among other things, methods ofidentifying a compound which, when complexed to a presenter protein,modulates a biological activity of a target protein. In someembodiments, such a method comprises contacting at least one TestCompound with a presenter protein and with a target protein, andassessing to what extent the Test Compound modulates (e.g., enhances orinterferes with) formation of a complex comprising both the presenterprotein and the target protein.

In general, a Test Compound has a structure that comprises bothPresenter Interacting Sites and Target Interacting Sites. Together, thePresenter Interacting Sites mediate binding between the Test Compoundand the presenter protein by making contacts with correspondinginteracting sites on the presenter protein; the Target Interacting sitesmediate binding between the Test Compound and the target protein bymaking contacts with corresponding interacting sites on the targetprotein. In some embodiments, the presenter and target proteins make oneor more direct contacts with one another.

In some embodiments, provided methods comprise the steps of:

-   -   a) Contacting a Test Compound with a presenter protein under        conditions suitable for formation of a binary complex comprising        the Test Compound and the presenter protein,    -   b) Contacting a binary complex with a target protein,    -   c) Assessing extent of formation and/or stability of a complex        comprising both the presenter protein and the target protein        when the Test Compound is present as compared to when it is        absent, and determining whether presence of the Test Compound        alters such extent of formation and/or stability.

In some embodiments, there is no detectable formation of a complexcomprising both a presenter protein and a target protein in the absenceof a Test Compound. In some embodiments, the amount and/or stability ofsuch complex formation is increased in the presence of a Test Compound.In some embodiments, the amount and/or stability of such complexformation is decreased in the presence of a Test Compound.

In some embodiments, a target protein has greater affinity for a binarycomplex relative to the affinity of a target protein for a presenterprotein in the absence of a binary complex. An increase in the affinityof a binary complex for a target protein indicates that a Test Compoundcan modulate the activity of a target protein. In some embodiments, abinary complex antagonizes the activity of a target protein. In someembodiments, a binary complex agonizes the activity of a target protein.

In some embodiments, provided such methods can be used to identify acompound which when complexed to a presenter protein modulates abiological activity of a target protein. In some embodiments, providedsuch methods can be used to identify a target protein whose biologicalactivity is modulated by a Test Compound.

Another embodiment of the invention is a method of developing a drugcandidate with modified selectivity towards a first target proteinrelative to a second target protein from a modulator compound thatmodulates the activity of both the first and second target protein. Themethod comprises the following steps:

-   -   a) providing a ligand compound and a presenter protein, wherein:        -   i) the ligand compound comprises a Presenter Interacting            Moiety that binds to the presenter protein to form a binary            complex; and a Target Interacting Moiety which enhances the            affinity of the binary complex for a first target protein            relative to the affinity of the presenter protein for the            first target protein in the absence of the binary complex;            and        -   ii) the binary complex does not substantially bind to a            second target protein;    -   b) covalently associating a modulator compound with the Target        Interacting Moiety of the ligand compound to form a modified        ligand compound;    -   c) contacting the modified ligand compound with the presenter        protein under conditions that allow the presenter protein to        bind to the modified ligand compound to form a modified binary        complex;    -   d) measuring the activity of the first target protein in the        presence of the modified binary complex and measuring the        activity of the first target protein in the presence of the        modulator compound;    -   e) measuring the activity of the second target protein in the        presence of the modified binary complex and measuring the        activity of the second target protein in the presence of the        modulator compound;    -   f) comparing the activity of the first and second target protein        in the presence of the modified binary complex and comparing to        the activity of the first and second target protein in the        presence of the modulator compound to assess whether the        modified binary complex has altered selectivity towards the        first target protein relative to the second target protein        compared with a modulator compound.

Yet another embodiment of the invention is a method of creating amodulator-based compound having modified selectivity towards a firsttarget protein relative to a second target protein, from a modulatorthat modulates the activity of both a first and second target protein.The method comprises the following steps:

-   -   a) providing a ligand compound, wherein:        -   i) a ligand compound comprises a Presenter Interacting            Moiety that binds to a presenter protein to form a binary            complex; and a Target Interacting Moiety which enhances the            affinity of the binary complex for a first target protein            relative to the affinity of the presenter protein for the            first target protein in the absence of the binary complex;            and        -   ii) the binary complex does not substantially bind to a            second target protein; and    -   b) covalently associating the modulator compound with the Target        Interacting Moiety of a ligand compound thereby creating a        modulator-based compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing affinity purification of protein complexesfollowed by mass spectrometric analysis allowing for targetidentification.

FIG. 2 is a schematic showing another representation of an affinitypurification of protein complexes followed by mass spectrometricanalysis allowing for target identification, using a FKBP/cyclophilinaffinity matrix.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

Unless otherwise indicated, the terms used herein are defined in thefollowing paragraphs.

The term “halo” or “halogen” refers to any radical of fluorine,chlorine, bromine or iodine.

The term “alkyl” refers to a monovalent hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₁₂ alkyl indicates that the group mayhave from 1 to 12 (inclusive) carbon atoms in it. In certain aspects,the term “alkyl” refers to a monovalent hydrocarbon chain that may be astraight chain or branched chain, containing 1 to 6 carbon atoms. Inother aspects, the term “alkyl” refers to a monovalent hydrocarbon chainthat may be a straight chain or branched chain, containing 1 to 4 carbonatoms.

The term “haloalkyl” refers to an alkyl in which one or more hydrogenatoms are replaced by halo, and includes alkyl moieties in which allhydrogens have been replaced by halo (e.g., perfluoroalkyl).

The term “alkenyl” refers to a monovalent straight or branchedhydrocarbon chain containing 2-20 carbon atoms and having one or moredouble bonds. Examples of alkenyl groups include, but are not limitedto, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. One ofthe double bond carbons may optionally be the point of attachment of thealkenyl substituent. In certain aspects, the term “alkenyl” refers to amonovalent straight or branched hydrocarbon chain containing 2-6 carbonatoms and having one or more double bonds. In other aspects, the term“alkenyl” refers to a monovalent straight or branched hydrocarbon chaincontaining 2-4 carbon atoms and having one or more double bonds.

The term “alkynyl” refers to a monovalent straight or branchedhydrocarbon chain containing 2-20 carbon atoms and characterized inhaving one or more triple bonds. Examples of alkynyl groups include, butare not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triplebond carbons may optionally be the point of attachment of the alkynylsubstituent.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and—NH(alkyl)₂ radicals respectively.

The term “aralkylamino” refers to a —NH(aralkyl) radical.

The term “alkylaminoalkyl” refers to a (alkyl)NH-alkyl-radical.

The term “dialkylaminoalkyl” refers to a (alkyl)₂N-alkyl-radical.

The term “mercapto” refers to an —SH radical.

The term “thioalkoxy” refers to an —S-alkyl radical.

The term “thioaryloxy” refers to an —S-aryl radical.

The term “alkoxy” refers to an —O-alkyl radical.

The term “aryl” refers to a monocyclic, bicyclic, or tricyclic aromatichydrocarbon ring system. Examples of aryl moieties include, but are notlimited to, phenyl, naphthyl, and anthracenyl.

The terms “arylalkyl” or “aralkyl” refer to an alkyl moiety in which analkyl hydrogen atom is replaced by an aryl group. Aralkyl includesgroups in which more than one hydrogen atom has been replaced by an arylgroup. Examples of “arylalkyl” or “aralkyl” include benzyl,2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and tritylgroups.

The term “carbocyclyl” refers to a non-aromatic, monocyclic, bicyclic,or tricyclic hydrocarbon ring system. Carbocyclyl groups include fullysaturated ring systems (e.g., cycloalkyls), and partially saturated ringsystems.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons. Any ring atom can be substituted (e.g., by one or moresubstituents). Examples of cycloalkyl moieties include, but are notlimited to, cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, andnorbornyl.

The term “heteroaryl” refers to a fully aromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatomsselected independently from N, O, or S if monocyclic, bicyclic, ortricyclic, respectively). Exemplary heteroaryl groups include furanyl,imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazolyl, pyrrolyl,pyridyl, pyrimidinyl, pyridazinyl, thiazolyl, triazolyl, tetrazolyl,thienyl, pyrimidinyl, pyridinyl, pyridazinyl carbazolyl, benzimidazolyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl,benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indolyl,isoindolyl, acridinyl, or benzisoxazolyl.

The term “heterocyclyl” refers to a nonaromatic, 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Theheteroatom may optionally be the point of attachment of the heterocyclylsubstituent. Examples of heterocyclyl include, but are not limited to,tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl, pyrimidinyl, and pyrrolidinyl.

Bicyclic and tricyclic ring systems containing one or more heteroatomsand both aromatic and non-aromatic rings are considered to beheterocyclyl groups according to the present definition. Such bicyclicor tricyclic ring systems may be alternately characterized as being anaryl or a heteroaryl fused to a carbocyclyl or heterocyclyl,particularly in those instances where the ring bound to the rest of themolecule is required to be aromatic.

The terms “heteroarylalkyl” and “heteroaralkyl”, as used herein, refersto an alkyl group substituted with a heteroaryl group.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocyclyl group.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted (e.g., by one or moresubstituents).

Unless otherwise specified, an optionally substituted ring system (i.e,aryl, heteroaryl, carbocyclyl, cycloalkyl, heterocyclyl, etc.) or ringsystem portions of a group (e.g., the aryl portion of an aralkyl group)may be substituted at one or more substitutable carbon atoms withsubstituents independently selected from: halo, —C≡N, C₁-C₄ alkyl, ═O,C₃-C₇ cycloalkyl, C₁-C₄ alkyl, —OH, —O—(C₁-C₄ alkyl)-, —SH, —S—(C₁-C₄alkyl), —(C₁-C₄ alkyl)-N(R^(b))(R^(b)), —N(R^(b))(R^(b)), —O—(C₁-C₄alkyl)-N(R^(b))(R^(b)), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R^(b))(R^(b)),—C(O)—N(R^(b))(R^(b)), —(C₁-C₄ alkyl)-C(O)—N(R^(b))(R^(b)),—O-(heteroaryl), —O-(heterocycle), —O-phenyl, -heteroaryl, -heterocycle,and -phenyl, wherein:

-   -   each R^(b) is independently selected from hydrogen, and —C₁-C₄        alkyl; or    -   two R^(b) are taken together with the nitrogen atom to which        they are bound to form a 4- to 8-membered saturated heterocycle        optionally comprising one additional heteroatom selected from N,        S, S(═O), S(═O)₂, and O,    -   any alkyl substituent is optionally further substituted with one        or more of —OH, —O—(C₁-C₄ alkyl), halo, —NH₂, —NH(C₁-C₄ alkyl),        or —N(C₁-C₄ alkyl)₂; and    -   any carbon atom on a phenyl, cycloalkyl, heteroaryl or        heterocycle substituent is optionally further substituted with        one or more of —(C₁-C₄ alkyl), —(C₁-C₄ fluoroalkyl), —OH,        —O—(C₁-C₄ alkyl), —O—(C₁-C₄ fluoroalkyl), halo, —NH₂, —NH(C₁-C₄        alkyl), or —N(C₁-C₄ alkyl)₂;

Unless otherwise specified, all optionally substituted heterocyclyl ringsystems (and any heterocyclyl substituents on any ring system) may besubstituted on any substitutable nitrogen atom with —C₁-C₄ alkyl, orfluoro-substituted C₁-C₄ alkyl.

The term “substituted” refers to the replacement of a hydrogen atom byanother group.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

Many proteins function by binding to a large number of differentpartners. Many of these proteins adapt the inherent variability of thehot spot protein regions to present the same residues in differentstructural contexts. More specifically, the protein-protein interactionscan be mediated by a class of natural products produced by a selectgroup of fungal and bacterial species. These molecules, referred toherein as “naturally occurring Presentation Ligands”, exhibit both acommon structural organization and resultant functionality that providesthe ability to modulate protein-protein interaction. These moleculescontain a First Region that is highly conserved and a Second Region thatexhibits a high degree of variability among the different naturallyoccurring Presentation Ligands. The constant region confers specificityfor the presenter protein and allows a naturally occurring PresentationLigand to bind to a presenter protein to form a binary complex; avariable region confers specificity for a target protein and allows abinary complex to bind to a target protein, typically modulating itsactivity.

Such naturally occurring Presentation Ligands are presented by presenterproteins, such as FKBPs and cyclophilins and act as diffusible,cell-penetrant, orally bio-available adaptors for protein-proteininteractions. Examples include well known and clinically relevantmolecules such as Rapamycin (Sirolimus), FK506 (Tacrolimus), andCyclosporin. In brief, these molecules bind endogenous intracellularpresenter proteins, the FKBPs e.g. rapamycin and FK506 or cyclophilinse.g. cyclosporin, and the resulting binary complexes of presenterprotein-bound Presentation Ligand selectively bind and inhibit theactivity of intracellular target proteins. Formation of a tripartitecomplex between the presenter protein, the Presentation Ligand and thetarget protein is driven by both protein-ligand and protein-proteininteractions and both are required for inhibition of the targetedprotein. In the example of the FKBP-rapamycin complex, the intracellulartarget is the serine-threonine kinase mTOR, whereas for FKBP-FK506complex, the intracellular target is the phosphatase calcineurin. Ofparticular interest in the preceding two examples, FKBP12 is utilized asa partner presentation protein by both the rapamycin and FK506presentation ligands. Moreover, the sub-structure components ofrapamycin and FK506 responsible for binding to FKBP12 are closelyrelated structurally, i.e. the so-called “Conserved Region,” but it isthe dramatic structural differences between rapamycin and FK506 in thenon FKBP12-binding regions, i.e. the “Variable Region,” that results inthe specific targeting of two distinct intracellular proteins, mTOR andcalcineurin, respectively. In this fashion, the Variable Regions ofrapamycin and FK506 are serving as contributors to the binding energynecessary for enabling presenter protein-target protein interaction.

Disclosed herein are methods of identifying new biologically activeagents having potential therapeutic utilities. These biologically activeagents act in an analogous manner to naturally occurring PresentationLigands by binding to presenter proteins, resulting in the formation ofa binary complex that binds with high affinity to new target proteins,thereby modulating their activity. In some embodiments, biologicallyactive agents identified by the disclosed assays “re-program” thebinding of presenter proteins to protein targets that either do notnormally bind to a presenter protein or greatly enhances bindingaffinity thereby resulting in the ability to modulate the activity ofthese new targets. As discussed above, naturally occurring PresentationLigands contain a first region that is highly conserved (referred toherein as the “Presenter Interacting Moiety” or “Conserved Region”) anda second region of variability (referred to herein as the “TargetInteracting Moiety” or “Variable Region”). A Presenter InteractingMoiety comprises one or more “Presenter Interacting Sites” that mediatebinding between the Test Compound and the presenter protein by makingcontacts with corresponding interacting sites on the presenter protein.A Target Interacting Moiety comprises one or more “Target InteractingSites” that mediate binding between the Test Compound and the targetprotein by making contacts with corresponding interacting sites on thetarget protein.

Presentation Ligands bind to presenter proteins at the Conserved Regionand, after formation of a binary complex with a presenter protein, bindto a target protein at the Variable Region. As such, a TargetInteracting Moiety of a naturally occurring Presentation Ligand could bevaried in many cases without significantly impairing the ability of aPresenter Interacting Moiety to bind to its presenter protein and form abinary complex. However, structural variation(s) in a Target InteractingMoiety combined with binding of a Presenter Interacting Moiety of anaturally occurring Presentation Ligand to presenter protein can alterthe binding specificity of the presenter protein/modified PresentationLigand binary complex, allowing it to bind to and alter the activity ofnew target proteins.

The methods disclosed herein are directed to assays in which binarycomplexes are screened against therapeutic targets. Binary complexescomprise a presenter protein and an analog of a naturally occurringPresentation Ligand. New target proteins are selected for which adesirable therapeutic effect can be achieved through modulation of itsbiological activity. Structural analogues of a naturally occurringPresentation Ligand, referred to herein as “Test Compounds” are thenprepared and/or selected. These structural analogues maintain theConserved Region of the naturally occurring Presentation Ligand or, atmost, contain insubstantial modifications that do not substantiallyimpair binding to the presenter protein. Structural modifications,however, are made to the Target Interacting Moiety with the objective ofreprogramming the binding specificity of a binary complex comprising thepresenter protein and Test Compound. The presenter protein and the TestCompound are then combined under conditions suitable for formation ofthe binary complex, which is then screened for its ability to bindand/or modulate the activity of new target proteins.

Suitable presenter proteins for use in the disclosed assays are thosewhich can bind a small molecule to form a binary complex, which canpotentially bind to and modulate the activity of a target protein. Insome embodiments, a presenter protein is one which is known to bind aPresentation Ligand to form a binary complex that is known to orsuspected of binding to and modulating the biological activity of atarget protein. Immunophilins are a class of presenter proteins whichare known to have these functions and include FKBPs and cyclophilins.

In certain embodiments, a presenter protein is an immunophilin. Incertain embodiments, a presenter protein is an immunophilin selectedfrom the group consisting of FKBPs and cyclophilins. Exemplary FKBPsinclude FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25,FKBP36, FKBP38, FKBP51, FKBP52, FKBP60 and FKBP65, to name but a few. Insome embodiments, a presenter protein is selected from the followingtable of FKBPs, which provides common names, an mRNA accession number, aprotein accession number, structural information, peptidyl prolineisomerase (PPI) activity, FK506 inhibition values, and rapamycininhibition values.

Protein mRNA accession Accession Amino FK506 Rapamycin Name Alias # #Acids MW PPI Inhibition Inhibition FKBP12 FKBP1A; FKBP12C NM_000801.3;P62942 108 12 yes  0.4 nM  0.2 nM NM_054014.2 FKBP12.6 FKBP1B; FKBP9NM_004116.3; P68106 108 12.6 yes  0.4 nM  0.2 nM NM_054033.1 FKBP13FKBP2 NM_001135208.1 P26885 142 13 yes   55 nM 1.14 NM_004470.3NM_057092.2 FKBP19 FKBP11 NM_016594.1; Q9NYL4 201 19 yes ″weak″ ndNM_001143781.1 NM_001143782.1 FKBP22 FKBP14 NM_017946.2 Q9NWM8 211 22 E.Nd nd Inferred FKBP23 FKBP7 NM_001135212.1 Q9Y680 259 23 E. Nd ndInferred FKBP25 FKBP3 NM_002013.2; Q00688 224 25 yes  160 nM  0.9 nMFKBP36 FKBP6 NM_001135211.1 Q91XW8 327 36 yes  400 nM   50 nM/0.9 nMNM_003602.3 FKBP38 FKBP8 NM_012181.3 Q14318 412 38 yes Nd  500 nM FKBP51FKBP54; FKBP5 NM_001145775.1 Q13451 457 51 yes yes, 14.7 nMNM_001145776.1 value NM_001145777.1 not NM_004117.3 reported FKBP52FKBP4; FKBP59 NM_002014.3 Q02790 459 59 yes   1 nM  1.4 nM/6 nM FKBP60|FKBP9; FKBP63 NM_007270; 095302 507 63 E. nd nd Inferred FKBP65 FKBP10NM_021939.2 Q96AY3 582 65 yes   45 nM 27-200 nM Terminology: PPI:peptidyl proline isomerase activity; E. Inferred: electronicllyinferred; nd: not determined as inferred by public domain search.

Exemplary cyclophilins include Cyp-A, PPIL1, PPIL3, USA-Cyp, Cyp-F,Cyp-B, Cyp-C, Cyp29, Cyp33, Cyp40, SDCCAG10, Cyp57, Cyp60, HAL539,Cyp88, NK-Cyp, and RanBP2, to name but a few. In some embodiments, apresenter protein is selected from the following table of cyclophilins,which provides common names, an mRNA accession number, a proteinaccession number, structural information, PPI activity, and cyclosporineinhibition values.

mRNA Protein Amino Cyclosporin Name Alias accession # Accession # AcidsMW PPI inhibition Cyp-A PP1A NM_021130 P62937 165 18 yes  6.8 nM PPIL1CYPL1, PP1B, NM_016059.4 Q9Y3C6 166 19 yes  9.8 nM CGI-124,UNQ2425/PRO4984 PPIL3 PPIL3; NM_032472.3 Q9H2H8-1; 161 23 nd ndCyclophilin J Q9H2H8-2 USA-Cyp CypH NM_006347 043447 177 20 yes  160 nMCyp-F Cyp3, PPIF NM_005729 P30405 207 22 yes  6.7 nM Cyp-BS-cyclophilin; SCYLP; NM_00942.2 P23284 216 24 yes  8.4 nM CYP-S1 Cyp-CPPIC NM_000943 P45877 212 23 yes  7.7 nm Cyp29 CAMLG; CAML NM_001745P49069 296 33 no nd Cyp33 CypE Q9UNP9 331 34 yes  6.9 nM Cyp40 CypD;PPID Q08752 370 40 yes   61 nM SDCCAG10 CWC27; NY-CO-10 NM_005869 Q6UX04472 54 no nd Cyp57 PPIL4 NM_139126 Q8WUA2 492 58 nd nd Cyp60 PPIL2NM_014337 Q13356-1; 520 59 no no Q1356-2 HAL539 Spliceosome-associatedNM_015342.2 Q96BP3 646 74 yes  168 nM cyclophilin Cyp88 Cyp-G NM_4792Q13427-1; 754 89 yes   51 nM Q13427-2 NK-Cyp NK-TR protein NM_005385P30414 1462 165 yes  488 nM RanBP2 NUP358 NM_006267 P49792 3224 360 nono Terminology: PPI: peptidyl proline isomerase activity; E. Inferred:electroniclly inferred; nd: not determined as inferred by public domainsearch.

It will be appreciated that presentation proteins used in accordancewith the present invention include allelic variants and splice variantsof the FKBPs and cyclophilins recited above.

As with a naturally occurring Presentation Ligand, a Test Compoundcomprises a Presenter Interacting Moiety and a Target InteractingMoiety. In some embodiments, a Test Compound consists of a PresenterInteracting Moiety and a Target Interacting Moiety. Although a TestCompound can be linear, it is preferred that a Test Compound is cyclic,e.g., a macrocycle with at least ten ring atoms, and more commonlybetween 10 and 40 ring atoms, and even more commonly between 18 and 30ring atoms. The ring atoms are typically selected from oxygen, nitrogen,carbon, sulfur and phosphorus and can unsubstituted or substituted andexist at various oxidation states.

In some embodiments, Presenter Interacting Sites and Target InteractingSites are located in discrete regions of a macrocyclic ring structure,e.g., that do not overlap, which can be represented schematically as:

wherein “TIS” is a Target Interacting Site, “PIS” is a PresenterInteracting Site, and L is a bond or a bivalent substituted orunsubstituted portion of a macrocyclic chain.

In some embodiments, Presenter and Target Interacting Sites areconnected to one another by a portion of a macrocyclic chain, which canbe represented schematically as:

wherein “TIS” is a Target Interacting Site, “PIS” is a PresenterInteracting Site, and L is a bond or a bivalent substituted orunsubstituted portion of a macrocyclic chain.

In some embodiments, Presenter Interacting Sites and Target InteractingSites are distributed on the Test Compound such that they interdigitatewith one another, which can be represented schematically as:

wherein “TIS” is a Target Interacting Site, “PIS” is a PresenterInteracting Site, and L is a bond or a bivalent substituted orunsubstituted portion of a macrocyclic chain. wherein each L isindependently selected from a bond or a bivalent macrocyclic chain.

It will be appreciated that the preceding three schematics arerepresentative structures that conceptually illustrate certainembodiments of the invention, and Test Compounds can have any number ofTarget Interacting Sites or Presenter Interacting Sites distributedthroughout the compound.

When L is a bivalent macrocyclic chain, it should be understood that allor a portion of one or more bivalent macrocyclic chains may contributeto or enhance either a) the ability of the Presenter Interacting Moietyto bind to its respective Presenter Protein; and/or b) the ability ofthe Target Interacting Moiety to interact with the Target Protein.

In some embodiments, and as disclosed earlier in U.S. Ser. No.61/427,626, the Variable Region (or Target Interacting Moiety) is aportion of a macrocyclic ring structure. It will be appreciated that theTarget Interacting Moiety may be varied in a multitude of waysincluding, but not limited to, modifications in the macrocyclic backboneand appendages to the macrocyclic backbone. In some embodiments, aVariable Region as described herein does not include the appendage of acomplete known ligand via a linker to the macrocyclic backbone, whereina “complete known ligand” is the entire chemical structure of a ligandthat, by itself, is known to bind to a target protein.

In some embodiments, each bivalent macrocyclic chain is a linear chainof up to 10 atoms, independently selected from carbon, nitrogen, oxygen,sulfur or phosphorous atoms, wherein each atom in the chain isoptionally substituted with one or more substituents independentlyselected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo,bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino,dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio,arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, andsulfonyl, and wherein any two atoms in the chain may be taken togetherwith the substituents bound thereto to form an optionally substitutedcarbocyclic, heterocyclic, aryl or heteroaryl ring, wherein the ring maybe further optionally fused to one or more optionally substitutedcarbocyclic, heterocyclic, aryl or heteroaryl rings.

As discussed above, a Presenter Interacting Moiety is comprised of oneor more Presenter Interacting Sites. In some embodiments, one or morePresenter Interacting Sites is highly conserved among naturallyoccurring Presentation Ligands. In some embodiments, an entire PresenterInteraction Moiety is highly conserved among naturally occurringPresentation Ligands. In some embodiments, such highly conservedPresenter Interacting Moieties and/or Presenter Interacting Sites allowa Test Compound to bind to a presenter protein, thereby forming a binarycomplex.

In one embodiment, a Presenter Interacting Moiety binds to a FKBP andhas the formula A:

wherein:

-   -   J is hydrogen or (C₁-C₂) alkyl;    -   K is (C₁-C₄)-straight or branched alkyl, benzyl or        cyclohexylmethyl; or    -   wherein J and K may be taken together to form a 5-7 membered        heterocyclic ring which may contain an O, S, SO or SO₂        substituent therein;    -   the stereochemistry at carbon position 1 is R or S;    -   each L is independently selected from a bond and a linear chain        of up to 10 atoms, independently selected from carbon, nitrogen,        oxygen, sulfur or phosphorous atoms, wherein each atom in the        chain is optionally substituted with one or more substituents        independently selected from alkyl, alkenyl, alkynyl, aryl,        heteroaryl, chloro, iodo, bromo, fluoro, hydroxy, alkoxy,        aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino,        carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,        alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and        wherein any two atoms in the chain may be taken together with        the substituents bound thereto to form a ring, wherein the ring        may be further substituted and/or fused to one or more        optionally substituted carbocyclic, heterocyclic, aryl or        heteroaryl rings;    -   X is selected from —NH—, —N(alkyl)-, —O—, —C(O)—, —CHOH—, —CH═,        or —CH₂—;    -   Y is selected from —C(O)NH—, —C(O)N(alkyl)-, —C(O)O—,        —C(O)C(O)—, —C(O)CHOH—, —C(O)CH═, —C(O)CH₂—, and —S(O)₂;    -   represents a single or a double bond; and    -   the points of attachment to the rest of the compound are through        a terminus of each L.

In one embodiment of Formula A, Y is selected from —C(O)NH—,—C(O)N(alkyl)-, —C(O)O—, —C(O)C(O)—, —C(O)CHOH—, —C(O)CH═ and —C(O)CH₂—;and J and K are taken together to form a 5-7 membered heterocyclic ringof the formula:

having the indicated stereochemistry, where the squiggly lines representwhere the ring binds to the rest of the First Region.

Specific examples of Presenter Interacting Moieties of Formula A,include the Conserved Region of FK506 shown below as Structural FormulaI; and the Conserved Region of rapamycin and antascomicin shown below asStructural Formula II, where the points of attachment to the rest of thecompound are represented by the squiggly lines.

Another example of a Presenter Interacting Moiety of Formula A is theConserved Region of certain rapamycin analogues (see U.S. Pat. No.7,276,498, the entire teachings of which are incorporated herein byreference) which is shown below as Structural Formula (III):

wherein R⁴ and R^(4′) are (a) independently selected from among H, OH,O(C₁ to C₆ alkyl), O(substituted C₁ to C₆ alkyl), O(acyl), O(aryl),O(substituted aryl), and halogen; or (b) taken together to form a doublebond to O; R⁵ is selected from H, OH, and OCH₃; X is selected from—CH₂—, —CHOH— and C═O; R⁶ is H or OCH₃; n is 1 or 2, and the points ofattachment to the rest of the compound are represented by the squigglylines. For Formula III, an aryl group is a C₆-C₁₀ carbocyclic aromaticgroup; and an acyl group is —CO(C₁-C₆ alkyl).

Still other examples of a Presenter Interacting Moiety of Formula A areother groups which mimic the rapamycin Conserved Region and arerepresented below as Structural Formulas IV and V:

wherein each R is independently hydrogen or methyl, each X₁ isindependently CH or N; X is C═O, CHOH or CH₂; n is 1 or 2, and thepoints of attachment to the rest of the compound are represented by thesquiggly lines.

Still other examples of a Presenter Interacting Moiety of Formula A areother groups which can function as mimics of the rapamycin ConservedRegion and are represented below as formula VI. Compounds represented byformula VI and their preparation are disclosed in U.S. Pat. No.6,037,370, the entire teachings of which are incorporated herein byreference.

wherein:

-   -   A is O, NH, or N—(C₁-C₄ alkyl);    -   B is CHL-Ar, (C₁-C₆)-alkyl, (C₂-C₆)-alkenyl, (C₅-C₇)-cycloalkyl,        (C₅-C₇)-cycloalkenyl, Ar substituted (C₁-C₆)-alkyl,        (C₅-C₇)-cycloalkenyl substituted (C₂-C₆)-alkenyl, Ar substituted        (C₂-C₆)-alkenyl, or

-   -   L and Q are independently hydrogen, (C₁-C₆)-alkyl or        (C₂-C₆)-alkenyl;    -   T is Ar or substituted cyclohexyl with substituents at positions        3 and 4 which are independently selected from the group        consisting of hydrogen, hydroxyl, O—(C₁-C₄)-alkyl,        O—(C₂-C₄)-alkenyl and carbonyl;    -   Ar is selected from the group consisting of 1-naphthyl,        2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl, 3-pyridyl,        4-pyridyl and phenyl having one to three substituents which are        independently selected from the group consisting of hydrogen,        halo, hydroxyl, nitro, CF₃, (C₁-C₆)-alkyl, (C₂-C₆)-alkenyl,        O—(C₁-C₄)-alkyl, O—((C₂-C₄)-alkenyl), O-benzyl, O-phenyl, amino        and phenyl;    -   D is hydrogen or U; E is either oxygen or CH—U, provided that if        D is hydrogen, then E is CH—U or if E is oxygen then D is not        hydrogen;    -   each U is independently selected from O—(C₁-C₄)-alkyl,        O—((C₂-C₄)-alkenyl), (C1-C6)-alkyl, (C2-C6)-alkenyl,        (C5-C7)-cycloalkyl, (C5-C7)-cycloalkenyl substituted with        (C1-C4)-alkyl or (C2-C4)-alkenyl, 2-indolyl, 3-indolyl,        (C1-C4)-alkyl-Ar, (C2-C4)-alkenyl-Ar, or Ar; and    -   J is hydrogen or (C1-C2) alkyl; K is (C1-C4)-alkyl, benzyl or        cyclohexylmethyl; or wherein J and K may be taken together to        form a 5-7 membered heterocyclic ring which may contain an O, S,        SO or SO₂ substituent therein;    -   the stereochemistry at carbon position 1 is R or S;    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of B and a covalent bond to a        terminus of D.

More preferably for formula VI, A is oxygen; J is hydrogen or (C1-C2alkyl); K is (C1-C4)-alkyl, benzyl or cyclohexylmethyl; or J and K aretaken together to form pyrrolidyl or piperidyl; and the stereochemistryat carbon position 1 is S.

In the above preferred group wherein J and K are taken together to formpyrrolidyl or piperidyl and E is CH—U, U is preferablydimethylaminophenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl,nitrophenyl, furyl, indolyl, pyridyl, or methylenedioxyphenyl.

In the above preferred groups wherein J and K are taken together to formpyrrolidyl or piperidyl and E is oxygen:

-   -   B is preferably benzyl, naphthyl, tert-butyl,    -   E-3-phenyl-2-methyl-prop-2-enyl, E-3-(4-hydroxyphenyl)        2-methyl-prop-2-enyl,        E-3-[trans(4-hydroxycyclohexyl)]-2-methyl-prop-2-enyl,        cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl,        cyclopentylopropyl, E-3-(4-methoxyphenyl)-2-methyl-prop-2-enyl,        E-3-(3,4-dimethoxyphenyl)-2-methyl-prop-2-enyl or        E-3-[cis(4-hydroxycyclohexyl)]-2-methyl-prop-2-enyl; and    -   D is preferably phenyl, methoxyphenyl, cyclohexyl, ethyl,        methoxy, nitrobenzyl, thiophenyl, indolyl, furyl, pyridyl,        pyridyl-N-oxide, nitrophenyl, fluorophenyl, trimethoxyphenyl or        dimethoxyphenyl.

Yet other compounds of Structural Formula A, which can function as aPresenter Interacting Moiety are mimics of the rapamycin ConservedRegions are represented below as Formulas VII-VIII. Compoundsrepresented by formulas VII-VIII and their preparation are disclosed inU.S. Pat. No. 6,037,370, the entire teachings of which are incorporatedherein by reference.

wherein

-   -   A′ is CH₂, oxygen, NH or N—(C1-C4 alkyl);    -   B′ and W are independently:    -   (i) Ar′, (C1-C10)-alkyl, (C2-C10)-alkenyl, (C2-C10)-alkynyl,        (C5-C7)-cycloalkyl substituted (C1-C6)-alkyl, (C5-C7)-cycloalkyl        substituted (C2-C6)-alkenyl, (C5-C7)-cycloalkyl substituted        (C2-C6)-alkynyl, (C5-C7)-cycloalkenyl substituted (C1-C6)-alkyl,        (C5-C7)-cycloalkenyl substituted (C2-C6)-alkenyl,        (C5-C7)-cycloalkenyl substituted (C2-C6)-alkynyl, Ar′        substituted (C1-C6)-alkyl, Ar′ substituted (C2-C6)-alkenyl, or        Ar′ substituted (C2-C6)-alkynyl wherein, in each case, any one        of the CH₂ groups of said alkyl, alkenyl or alkynyl chains may        be optionally replaced by a heteroatom selected from the group        consisting of O, S, SO, SO₂, N, and NR, wherein R is selected        from the group consisting of hydrogen, (C1-C4)-alkyl,        (C2-C4)-alkenyl or alkynyl, and (C1-C4) bridging alkyl wherein a        bridge is formed between the nitrogen and a carbon atom of said        heteroatom-containing chain to form a ring, and wherein said        ring is optionally fused to an Ar′ group; or

wherein Q′ is hydrogen, (C1-C6)-alkyl or (C2-C6)-alkenyl or alkynyl;

-   -   T′ is Ar′ or substituted 5-7 membered cycloalkyl with        substituents at positions 3 and 4 which are independently        selected from the group consisting of oxo, hydrogen, hydroxyl,        O—(C1-C4)-alkyl, and O—(C2-C4)-alkenyl;    -   Ar′ is a carbocyclic aromatic group selected from the group        consisting of phenyl, 1-naphthyl, 2-naphthyl, indenyl, azulenyl,        fluorenyl, and anthracenyl; or a heterocyclic aromatic group        selected from the group consisting of 2-furyl, 3-furyl,        2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrrolyl,        oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,        isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl,        1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl,        1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl,        isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl,        benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl,        purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl,        phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,        pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,        and phenoxazinyl;    -   Ar′ may contain one to three substituents which are        independently selected from the group consisting of hydrogen,        halogen, hydroxyl, hydroxymethyl, nitro, trifluoromethyl,        trifluoromethoxy, (C1-C6)-alkyl, (C2-C6)-alkenyl,        O—(C1-C4)-alkyl, O—(C2-C4)-alkenyl, O-benzyl, O-phenyl,        1,2-methylenedioxy, amino, carboxyl, N—[(C1-C5)-alkyl or        (C2-C5)-alkenyl)carboxamides, N,N-di-[(C1-C5)-alkyl or        (C2-C5)-alkenyl)]carboxamides, N-morpholinocarboxamide,        N-benzylcarboxamide, N-thiomorpholinocarboxamide,        N-picolinoylcarboxamide, O—X, CH₂—(CH₂)_(q)X, O—(CH₂)_(q)X,        (CH₂)_(q)O—X, and CH═CH—X;    -   X is 4-methoxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrazyl,        quinolyl, 3,5-dimethylisoxazoyl, isoxazoyl, 2-methylthiazoyl,        thiazoyl, 2-thienyl, 3-thienyl, or pyrimidyl;    -   q is 0-2;    -   G is hydrogen or U′;    -   M is either oxygen or CH—U′; provided that if G is hydrogen,        then M is CH—U′ or if M is oxygen, then G is U′;    -   U′ is O—[(C1-C4)-alkyl]. O—[(C2-C4)-alkenyl], (C1-C6)-alkyl,        (C2-C6)-alkenyl, (C5-C7)-cycloalkyl, (C5-C7)-cycloalkenyl        substituted with (C1-C4)-alkyl or (C2-C4)-alkenyl,        (C1-C4)-alkyl-Y, (C2-C4)-alkenyl-Y or Y;    -   Y is selected from the group consisting of phenyl, 1-naphthyl,        2-naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl,        2-pyrrolinyl, 3-pyrrolinyl, pyrolidinyl, 1,3-dioxolyl,        2-imidazolinyl, imidazolidinyl, 2H-pyranyl, 4H-pyranyl,        piperidyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,        thiomorpholinyl, piperazinyl, quinuclidinyl, and heterocyclic        aromatic groups as defined for Ar′ above;    -   Y may contain one to three substituents which are independently        selected from the group consisting of hydrogen, halogen,        hydroxyl, hydroxymethyl, nitro, trifluoromethyl,        trifluoromethoxy, (C1-C6)-alkyl, (C2-C6)-alkenyl,        O—(C1-C4)-alkyl, O—(C2-C4)-alkenyl, O-benzyl, O-phenyl,        1,2-methylenedioxy, amino, and carboxyl;    -   J′ is hydrogen, (C1-C2) alkyl or benzyl;    -   K′ is (C1-C4)-alkyl, benzyl or cyclohexylmethyl, or wherein J′        and K may be taken together to form a 5-7 membered heterocyclic        ring which may contain a heteroatom selected from the group        consisting of O, S, SO and SO₂; and    -   n is 1 or 2;    -   m is an integer from 0-10, preferably 0-3; and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of B′ and a covalent bond to a        terminus of G for Formula VII or a covalent bond to Ar′ for        formula VIII.

Preferably, B′ and W are independently selected from:

Ar′, (C1-C10)-alkyl, (C2-C10)-alkenyl, (C2-C10)-alkynyl,(C5-C7)-cycloalkyl substituted (C1-C6)-alkyl, (C5-C7)-cycloalkylsubstituted (C2-C6)-alkenyl, (C5-C7)-cycloalkyl substituted(C2-C6)-alkynyl, (C5-C7)-cycloalkenyl substituted (C1-C6)-alkyl,(C5-C7)-cycloalkenyl substituted (C2-C6)-alkenyl, (C5-C7)-cycloalkenylsubstituted (C2-C6)-alkynyl, Ar′ substituted (C1-C6)-alkyl, Ar′substituted (C2-C6)-alkenyl or Ar′ substituted (C2-C6)-alkynyl wherein,in each case, any one of the CH₂ groups of said alkyl, alkenyl oralkynyl chains may be optionally replaced by a heteroatom selected fromthe group consisting of O, S, SO, SO₂; or

wherein

-   -   any Ar′ may contain one to three substituents which are        independently selected from the group consisting of hydrogen,        halogen, hydroxyl, hydroxymethyl, nitro, trifluoromethyl,        trifluoromethoxy, (C1-C6)-alkyl, (C2-C6)-alkenyl,        O—[(C1-C4)-alkyl], O—[(C2-C4)-alkenyl], O-benzyl, O-phenyl,        1,2-methylenedioxy, amino and carboxyl; and    -   Y is selected from the group consisting of phenyl, 1-naphthyl,        2-naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl and        heterocyclic aromatic groups as defined for Ar′ above.

In another preferred embodiment for Formula VII and VIII, at least oneof B′ or W is independently selected from the group consisting of(C2-C10)-alkynyl; (C5-C7)-cycloalkyl substituted (C2-C6)-alkynyl;(C5-C7)-cycloalkenyl substituted (C2-C6)-alkynyl; and Ar′ substituted(C2-C6)-alkynyl.

Alternatively, at least one of B′ or W is independently represented bythe formula —(CH₂)_(r)—(Z)—(CH₂)_(s)Ar′, wherein:

-   -   Z is independently selected from the group consisting of CH₂, O,        S, SO, SO₂, N, and NR;    -   r is 0-4;    -   s is 0-1; and    -   Ar′ and R are as defined above in formula VII.

In yet another alternative embodiment of formula VII or VIII, at leastone of B′ or W is independently selected from the group consisting ofAr′, Ar′-substituted (C1-C6)-alkyl, Ar′-substituted (C2-C6)-alkenyl andAr′-substituted (C2-C6)-alkynyl;

-   -   wherein Ar′ is substituted with one to three substituents which        are independently selected from the group consisting of        N—((C1-C5)-alkyl or (C2-C5)-alkenyl) carboxamides,        N,N-di-((C1-C5)-alkyl or (C2-C5)-alkenyl)carboxamides,        N-morpholinocarboxamide, N-benzylcarboxamide,        N-thiomorpholinocarboxamide, N-picolinoylcarboxamide, O—X,        CH₂—(CH₂)_(q)—X, O—(CH₂)_(q)—X, (CH₂)_(q)O—X, and CH═CH—X; and        Ar′, X and q are as defined above.

Specific Conserved Regions are disclosed in U.S. Pat. No. 6,037,370 andare contemplated for use as First Regions in the disclosed methods.

Yet another embodiment of Formula A which can serve as a PresenterInteracting Moiety in the present invention is disclosed in U.S. Pat.No. 5,935,954 (the entire teachings of which are incorporated herein byreference) and represented by formula (IX):

wherein:

-   -   A is CH₂, O, NH or N—[(C1-C4)-alkyl];    -   B is (C1-C6)-alkyl, (C2-C6)-alkenyl or (C2-C6)-alkynyl, wherein        one of the carbon atoms of B is optionally replaced by O, S, SO,        SO₂, NH or N—[(C1-C4)-alkyl];    -   D is 1-[(C1-C4)-alkyl]-4-piperidinyl; 1-piperazinyl;        1-[(C1-C4)-alkyl]-4-piperazinyl; a 5-7-membered cycloalkyl or        cycloalkenyl ring optionally comprising substituents at the 3        and/or 4 position of said ring, wherein said substituents are        selected from oxo, OH, (C1-C4)-alkyl, O—(C1-C4)-alkyl,        O—(C2-C4)-alkenyl, NH₂, N,N di-[(C1-C4)-alkyl]amino or halogen;        or a monocyclic or bicyclic aromatic ring structure consisting        of 5 to 6 members in each ring and optionally comprising up to 4        heteroatoms independently selected from N, O or S;    -   E is SO₂ or —C(O)—C(O)—;    -   G is 1-[(C1-C4)-alkyl]-4-piperidinyl, 1-piperazinyl,        1-[(C1-C4)-alkyl]-4-piperazinyl, (C1-C7)-alkyl, (C2-C7)-alkenyl,        (C2-C7)-alkynyl, (C5-C7)-cycloalkyl, or a monocyclic or bicyclic        aromatic ring structure consisting of 5 to 6 members in each        ring; wherein up to two carbon atoms in any G are optionally and        independently replaced by O, S, SO, SO₂ or N;    -   G optionally comprises up to three substituents independently        selected from halogen, hydroxyl, (C1-C6)-alkyl, (C2-C6)-alkenyl,        O—(C1-C5)-alkyl, O—(C2-C5)-alkenyl, O-benzyl, amino, carboxyl,        N—[(C1-C5)-alkyl], N—[(C2-C5)-alkenyl], trifluoromethyl or        trifluoromethoxy; and wherein one carbon atom of any individual        substituent is optionally replaced by O, N or S;    -   Q is a five membered aromatic ring containing 1 to 2 heteroatoms        selected from N, O or S, or a six membered aromatic ring        containing 0 to 2 heteroatoms selected from N, O or S;    -   J is a monocyclic or bicyclic aromatic ring structure attached        to the 3 position of Q consisting of 5 to 6 members in each        ring, optionally comprising up to four heteroatoms independently        selected from O, S, or N; and    -   J optionally comprises up to 3 substituents independently        selected from halo, OH, CH₂OH, NO₂, SO₃H, trifluoromethyl,        trifluoromethoxy, O-phenyl, 1,2-methylenedioxy, NR₁R₂, amino,        carboxyl, N—[(C1-C5)-alkyl]-carboxamide,        N—[(C2-C5)-alkenyl]-carboxamide, N-morpholinocarboxamide,        N-benzylcarboxamide, N-thiomorpholinocarboxamide,        N-picolinoylcarboxamide, morpholinyl, piperidinyl, O—R₃,        CH₂—(CH₂)_(q)R₃, O—(CH₂)_(q)R₃, (CH₂)_(q)O—R₃, CH═CH—R₃,        (C1-C6)-alkyl, or (C2-C6)-alkenyl, wherein in any substituent        one carbon atom is optionally replaced by a heteroatom selected        from the group consisting of O, S, SO, SO₂, NH or        N—[(C1-C4)-alkyl];    -   wherein R₁ and R₂ are independently selected from the group        consisting of hydrogen, (C1-C6)-alkyl, (C2-C6)-alkenyl,        (C2-C6)-alkynyl and benzyl;    -   R₃ is selected from the group consisting of 4-methoxyphenyl,        2-pyridyl, 3-pyridyl, 4-pyridyl, pyrazyl, quinolyl,        3,5-dimethylisoxazoyl, 2-methylthiazoyl, thiazoyl, 2-thienyl,        3-thienyl and pyrimidyl; and    -   q is 0-2;    -   n is 1 or 2;    -   the stereochemistry at each of “1” and “2” is independently        selected from (R) and (S); and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of J and a covalent bond to a        terminus of G.

The “3 position of Q” recited above is relative to the point ofattachment of Q to the rest of the compound. For the purposes of thisapplication, this point of attachment is designated the 1 position,regardless of any potential conflict with accepted chemicalnomenclature.

Another embodiment of Formula A which can serve as a PresenterInteracting Moiety in the present invention is also disclosed in U.S.Pat. No. 5,935,954 and is represented by formula (X):

wherein:

-   -   A, B, D, E, G and Q are as defined as above for Formula IX;    -   K is H, (C5-C7) cycloalkyl, (C5-C6) aromatic ring,        1-[(C1-C4)-alkyl]-4-piperidinyl, 1-piperazinyl,        1-[(C1-C4)-alkyl]-4-piperazinyl, (C1-C7)-alkyl, (C2-C7)-alkenyl        or (C2-C7)-alkynyl, wherein up to two carbon atoms in K are        optionally replaced independently by O, S, SO, SO₂, NH, NO or        N—(C1-C4)-alkyl,    -   wherein K optionally comprises up to 2 substituents        independently selected from halo, amino, hydroxy, carboxy,        methoxy or (C1-C3)alkyl; and    -   L and M are independently selected from H, (C1-C7)-alkyl,        (C2-C7)-alkenyl or (C2-C7)-alkynyl, wherein one carbon atom in        R₂ and R₃ is optionally replaced by O, S, SO, SO₂NH or        N—(C1-C4)-alkyl,    -   wherein L and M optionally comprise up to two substituents        independently selected from halogen, hydroxy, amino, carboxy, or        a 5 to 6 membered aromatic ring, said aromatic ring comprising        up to two heteroatoms selected from N, O or S;    -   n is 1 or 2;    -   the stereochemistry at each of “1” and “2” is independently        selected from (R) and (S); and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of M and a covalent bond to a        terminus of G.

Preferably, none of the monocyclic or bicyclic rings that may be presentin either compounds of formulae (IX) or (X) contain more than oneheteroatom per ring.

More preferably, in compounds of formulae (IX) and (X), A is oxygen andE is —C(O)—C(O)—. Even more preferred are compounds wherein n is 2 andthe potential location of heteroatoms in Q exclude the 1 and 3 positions(i.e., the position where the aromatic ring is bound to the rest of themolecule and the position where J is bound to the aromatic ring). Thesecompounds are represented by formulae (XI) and (XII):

wherein:

-   -   B is propyl, ethyl, or 1-methylethenyl;    -   D is phenyl, N-morpholinyl, 4-hydroxy-cyclohexyl,        4-(N-methyl)-piperidinyl, 4-pyridyl or pyranyl;    -   G is 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl,        4-fluorophenyl, 2-furanyl, 1,1-dimethyl-2-methoxyethyl, t-butyl,        4-(4-hydroxy) pyranyl, isobutyl, 4-pyranyl, isobutyl, isopropyl,        1-methylcyclohexyl, 1,1,2-trimethylpropyl, 1-hydroxycyclohexyl,        1-trimethylpropyl, 4-methoxy-1-hydroxy-cyclohexyl,        5-methoxymethyl-2-methylphenyl, 2-methylcyclohexyl,        5-(1-methyl-1-methoxyethyl)-2-methylcyclohexyl-2-enyl,        2-methylcyclohexyl,        5-(1-methyl-1-methoxyethyl)-2-methylcyclohexyl,        5-ethoxy-2-methylcyclohexyl, 4-ethoxy-N-aceto-2-pyrrolidinyl, or        5-isopropyl-2-methylcyclohexyl; and    -   J is 4-phenyl-1-(3-pyridyl)-1-butenyl, 2,5-diethoxyphenyl,        4-phenyl-1-(3-pyridyl-N-oxide)-1-butenyl, 2-methoxyphenyl,        1-(3-pyridyl)-1-pentenyl, 2-ethoxyphenyl, 2,5-dipropoxyphenyl,        2,6-dimethoxyphenyl, 1-(3-pyridyl)-1-butenyl,        1-(3-pyridyl)-1-pentenyl, 1-(3-pyridyl)-1-hexenyl,        1-(4-methylphenyl)-1-pentenyl, 2,6-dimethoxymethylphenyl,        1-cyclohexyl-1-pentenyl, 2-ethoxymethyl-N-indolyl,        1-cyclohexyl-3-methoxy-1-propenyl, 2,6-diethoxymethylphenyl,        1-(3-pyridyl)-1-hexa-1,5-dienyl,        1-(4-pyranyl)-1-hexa-1,5-dienyl, 1-cyclohexyl-1-hexenyl,        2,5-dipropyl-N-pyrrolyl, 2-methyl-5-butyl-N-pyrrolyl,        3-(1-methoxy)-2-hexenyl, 3-(1-methoxy)-4-methyl-2-pentenyl,        2,5-dimethyl-N-pyrrolyl, 3-(2-methyl)-3-heptenyl or        2-(2-hexenyl);    -   W, X, Y and Z are independently selected from CH, N, O or S;    -   the stereochemistry at each of “1” and “2” is independently        selected from (R) and (S); and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of J and a covalent bond to a        terminus of G.

For formulas IX-XII, exemplary aromatic groups include phenyl, naphthyl,furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazolyl,pyrrolyl, pyridyl, pyrimidinyl, pyridazinyl, thiazolyl, triazolyl,tetrazolyl, thienyl, pyrimidinyl, pyridinyl, pyridazinyl carbazolyl,benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl,benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl,isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzisoxazolyl.

Individual compounds disclosed in U.S. Pat. No. 5,935,954 can also beused in the disclosed methods as Presenter Interacting Moieties ofFormula A.

In another embodiment, a Presenter Interacting Moiety binds to acyclophilin and is represented by Formula B:

wherein each L is independently selected from a bond and a linear chainof up to 10 atoms, independently selected from carbon, nitrogen, oxygen,sulfur or phosphorous atoms, wherein each atom in the chain isoptionally substituted with one or more substituents independentlyselected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo,bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino,dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio,arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, andsulfonyl, and wherein any two atoms in the chain may be taken togetherwith the substituents bound thereto to form a ring, wherein the ring maybe further substituted and/or fused to one or more optionallysubstituted carbocyclic, heterocyclic, aryl or heteroaryl rings;

-   -   each X is independently selected from —NH—, —N(alkyl)-, —O—,        —C(O)—, —CHOH—, or —CH₂—; and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of each L.

In a more specific embodiment of Formula C, a Presenter InteractingMoiety has the structure of the Conserved Region of cyclosporinerepresented by Structural Formula XIII:

where the point of attachment to the rest of the compound is representedby the squiggly lines (see WO2010/034243, the entire teachings of whichare incorporated herein by reference).

In another embodiment, a Presenter Interacting Moiety binds to acyclophilin and is represented by Formula C:

wherein

-   -   X and L are as defined for Formula (B);    -   R¹ is selected from (C1-C6)-alkyl, (C1-C6)-alkenyl,        (C1-C6)-alkynyl, aryl, (C3-C7)-carbocyclyl, —(C1-C4        alkylene)-aryl, and —(C1-C4 alkylene)—(C3-C7 carbocyclyl;    -   each R₂ is independently selected from halo, —C≡N, C1-C4 alkyl,        ═O, C3-C7 cycloalkyl, C1-C4 alkyl, —OH, —O—(C1-C4 alkyl)-, —SH,        —S—(C1-C4 alkyl), —(C1-C4 alkyl)-N(R^(b))(R^(b)),        —N(R^(b))(R^(b)), —O—(C1-C4 alkyl)-N(R^(b))(R^(b)), —(C1-C4        alkyl)-O—(C1-C4 alkyl)-N(R^(b))(R^(b)), —C(O)—N(R^(b))(R^(b)),        —(C1-C4 alkyl)-C(O)—N(R^(b))(R^(b)), —O-(heteroaryl),        —O-(heterocycle), —O-phenyl, -heteroaryl, -heterocycle, and        -phenyl, wherein:        -   each R^(b) is independently selected from hydrogen, and            —C1-C4 alkyl; or    -   two R^(b) are taken together with the nitrogen atom to which        they are bound to form a 4- to 8-membered saturated heterocycle        optionally comprising one additional heteroatom selected from N,        S, S(═O), S(═O)₂, and O,        -   any alkyl substituent is optionally further substituted with            one or more of —OH, —O—(C1-C4 alkyl), halo, —NH₂, —NH(C1-C4            alkyl), or —N(C1-C4 alkyl)₂; and        -   any carbon atom on a phenyl, cycloalkyl, heteroaryl or            heterocycle substituent is optionally further substituted            with one or more of —(C1-C4 alkyl), —(C1-C4 fluoroalkyl),            —OH, —O—(C1-C4 alkyl), —O—(C1-C4 fluoroalkyl), halo, —NH₂,            —NH(C1-C4 alkyl), or —N(C1-C4 alkyl)₂;    -   m is 0, 1, 2 or 3; and    -   the points of attachment to the rest of the compound is by a        covalent bond to a terminus of each L.

In a more specific embodiment of Formula C, a Presenter InteractingMoiety has the structure of the Conserved Region of sanglifehrinrepresented by Structural Formula XIV (see WO2010/034243):

wherein the points of attachment to the rest of the molecule arerepresented by the squiggly lines.

In certain embodiments, structural modifications can be made to formulasI-XIV, provided that they do not substantially reduce the ability of aPresenter Interacting Moiety to form a complex with a presenter protein,e.g., reduce binding by more than 5%, 10%, 15%, 20%, 25%, or 30%. Incertain embodiments, structural modifications can be made to formulasI-XIV, provided that they do not substantially reduce the ability of aPresenter Interacting Moiety to form a complex with an immunophilin,such as an FKBP or cyclophilin, by more than 5%, 10%, 15%, 20%, 25%, or30%. Reduction in binding between the Presenter Interacting Moiety andpresenter proteins can be determined by methods well known in the art,e.g., as described below in the subsequent paragraphs.

The Target Interacting Moiety, also referred to herein as the “VariableRegion”, is for enhancing the affinity of the binary complex for atarget relative to the affinity of a presenter protein alone (withoutthe binary complex) or a Test Compound alone (without the binarycomplex). In some embodiments, a presenter protein alone and/or a TestCompound alone will have no affinity (i.e., does not bind) for a targetprotein. In these instances, enhancement of affinity of a binary complexfor a target protein can be assessed merely by determining using methodsknown in the art, e.g., by mass spectrometry based proteomic strategy(e.g., for example Liang et al., Current Proteomics 6:25 (2009) andBauer and Kunster Eur. J. Biochem. 270:570 (2003), the entire teachingsof which are incorporated herein by reference), whether a ternarycomplex of a presenter protein, Test Compound, and target proteinformed. Alternatively, where there is some affinity between a targetprotein and a presenter protein and/or Test Compound, the actual bindingaffinities of each complex are measured by methods known in the art. Forexample, to ascertain whether affinity of a binary complex for a targetprotein is increased relative to either the affinity of a compound for atarget protein in the absence of a binary complex or affinity of apresenter protein in the absence of a binary complex, requirescalculation of the dissociation constant (K_(d)). The K_(d) is aspecific type of equilibrium constant that measures the propensity of alarger object to separate (dissociate) reversibly into smallercomponents, as when a complex falls apart into its component molecules.For example, the (K_(d)) describes the affinity between a ligand (L)(such as a drug) and a protein (P) i.e. how tightly a ligand binds to aparticular protein.

There are multiple established technical methods to calculate K_(d)(reviewed in Stockwell, B. R. Nature; 432 (7019): 846-54 (2004)) andhave applicability to generate data to address questions as highlightedabove. Specific techniques such as Surface Plasmon Resonance (SPR) andFluorescence Polarization (FP) may represent preferred but not exclusivemethodologies for calculating K_(d). SPR measures the change inrefractivity of a metal surface when a compound binds to a protein thatis immobilized on that metal surface (Homola, J., Annal. Bioanal. Chem.;377: 528-539 (2003)) while FP measures the change in tumbling rate for acompound when it is bound to a protein using loss-of-polarization ofincident light (Burke, T. J., et. al. Comb. Chem. High ThroughputScreen; 6: 183-194 (2003)). Each of these technologies has provenamenable to calculating the K_(d) of protein-drug-protein complexes(Banaszynski, L. A., et. al. J. Am. Chem. Soc.; 127: 4715-4721 (2005))and thus provides means to determine whether the affinity of theaforementioned binary complex for the target protein is increasedrelative to either the affinity of the compound for the target proteinin the absence of the binary complex or affinity of the presenterprotein in the absence of the binary complex.

In some embodiments, binding affinity is assessed by measuring whether abiological activity of a target protein is modulated by a binarycomplex. In some embodiments, the Variable Region of a macrocycliccompound is bivalent, i.e., has two ends, each of which covalently bindsto an end of the Conserved Region. This is depicted structurally belowin Structural Formulas A1, B1, C1 and Ia-XIVa:

The variables in Structural Formulas A1, B1, C1 and Ta through XIVa areas defined above and described in classes and subclasses herein (e.g.,corresponding Structural Formulas A, B, C and I-XIV).

In some embodiments, a Variable Region is a linear bivalent C4 to C30(preferably C6-C20, more preferably C6-C15) aliphatic group consistingof carbon and hydrogen, optionally comprising one or double bonds. Oneor more of the carbon atoms in the aliphatic group can be optionallyreplaced with a functional group selected from O, S, SO, SO₂, CO, COO,OCO, CONR², OCONR², NR², SO₂NR², NR²CONR² and NR²SO₂NR. The functionalgroup can be bidirectional (e.g., CONR² and NR²CO are both included) andR₂ is hydrogen or an alkyl group optionally substituted with one or moregroups selected from —CN, —NO₂, —OR^(c), —NR^(a)R^(b), —S(O)_(i)R^(c),—NR^(d)S(O)_(i)R^(c), —S(O)_(i)NR^(e)R^(f), —C(═O)OR^(c), —OC(═O)OR^(c),—C(═S)OR^(c), —O(C═S)R^(c), —C(═O)NR^(e)R^(f), —NR^(d)C(═O)R^(c),—C(═S)NR^(e)R^(f), —NR^(d)C(═S)R^(c), —NR^(d)(C═O)OR^(c),—O(C═O)NR^(e)R^(f), —NR^(d)(C═S)OR^(c), —O(C═S)NR^(e)R^(f),—NR^(d)(C═O)NR^(e)R^(f), —NR^(d)(C═S)NR^(e)R^(f), —C(═S)R^(c) and—C(═O)R^(c); R^(a) and R^(b) are each independently —H or (C1-C6)alkyloptionally substituted with 1 to 3 substituents independently selectedfrom the group consisting of halogen, hydroxy, —NR^(g)R^(h) and(C1-C3)alkoxy; R^(c) is —H, or (C1-C6)alkyl optionally substituted with1 to 3 substituents independently selected from the group consisting ofhalogen, hydroxy and (C1-C3)alkoxy; R^(d) is —H or (C1-C6)alkyloptionally substituted with 1 to 3 substituents independently selectedfrom the group consisting of halogen, hydroxy and (C1-C3)alkoxy; R^(e)and R^(f) are each independently —H or (C1-C6)alkyl optionallysubstituted with 1 to 3 substituents independently selected from thegroup consisting of halogen, hydroxy and (C1-C3)alkoxy; and R^(g) andR^(h) are each independently selected from the group consisting of —H,(C1-C6)alkyl, halo(C1-C6)alkyl, hydroxy(C1-C6)alkyl and(C1-C3)alkoxy(C1-C6)alkyl. Each carbon atom in the aliphatic group isoptionally substituted with —CN, —NO₂, —OR^(c), —NR^(a)R^(b),—S(O)_(i)R^(c), —NR^(d)S(O)_(i)R^(c), —S(O)_(i)NR^(e)R^(f), C(═O)OR,—OC(═O)OR^(c), —C(═S)OR^(c), —O(C═S)R^(c), —C(═O)NR^(e)R^(f),—NR^(d)C(═O)R^(c), —C(═S)NR^(e)R^(f), —NR^(d)C(═S)R^(c),—NR^(d)(C═O)OR^(c), —O(C═O)NR^(e)R^(f), —NR^(d)(C═S)OR^(c),—O(C═S)NR^(e)R^(f), —NR^(d)(C═O)NR^(e)R^(f), —NR^(d)(C═S)NR^(e)R^(f),—C(═S)R^(c) and —C(═O)R^(c). The bivalent aliphatic group can also besubstituted with a targeting group that mimics a ligand that binds to atarget protein, wherein the targeting group is less than the ligandknown to bind a target protein. In other words, a targeting group is atruncated portion or a fragment of a ligand. In some embodiments, atargeting group is a pharmacophore of a ligand. In some embodiments, atargeting group is a fragment of a pharmacophore of a ligand. Examplesof such targeting groups include phosphotyrosine mimics, ATP mimetics,and the like. While not wishing to be bound by any particular theory, itis believed that a truncated portion or fragment of a ligand will formsome contacts that contribute to the binding energy enabling presenterprotein-target protein interaction, but that additional contacts betweenthe target protein and one or more ring atoms in the Variable Regionwill also contribute to the binding energy. In some embodiments, atargeting group is one of multiple Target Interacting Sites in a TargetInteracting Moiety. Additional contacts between the target protein andpresenter protein may also contribute to the binding energy of thepresenter protein-target protein interaction.

In some embodiments, a Test Compound is a macrocycle and a targetprotein binds to a ring atom in the Variable Region (i.e., a ring atomof a Target Interacting Moiety). In some embodiments, a target proteinbinds to two or more ring atoms in a Variable Region. In someembodiments, a target protein binds to a substituent attached to one ormore ring atoms in a Variable Region. In some embodiments, a targetprotein binds to one or more ring atoms in the Variable Region and to asubstituent attached to one or more ring atoms in a Variable Region. Incertain embodiments, a target protein binds to a group that mimics aligand of a target protein and wherein the group that mimics a ligand ofa target protein is attached to a Variable Region. In certainembodiments, a target protein binds to a presenter protein and theaffinity of a target protein for a presenter protein in a binary complexis increased relative to the affinity of a target protein for apresenter protein in the absence of the complex. In some embodiments,binding in the preceding examples is through non-covalent interactionsof a target protein to a Variable Region.

A target protein is a protein which mediates a disease condition or asymptom of a disease condition. As such, a desirable therapeutic effectcan be achieved by modulating (inhibiting or increasing) its activity.Such desirable therapeutic effects include those where the activity of atarget protein is measured directly or indirectly (e.g., wheremodulating the target protein has a measureable effect on a downstreamprocess, substrate, activity, etc., which is more easily measured and/orobserved). In some embodiments, target proteins which are tested in anassay are those which do not naturally associate with a presenterprotein, e.g., those which have an affinity for a presenter protein inthe absence of a binary complex with a Presentation Ligand of greaterthan 1 μM, preferably greater than 5 μM and more preferably greater than10 μM. In some embodiments, target proteins which do not naturallyassociate with a presenter protein are those which have an affinity fora Test Compound in the absence of a binary complex with a PresentationLigand of greater than 1 μM, preferably greater than 5 μM and morepreferably greater than 10 μM. In certain embodiments, target proteinswhich do not naturally associate with a presenter protein are thosewhich have an affinity for a binary complex of cyclosporine, rapamycin,or FK506 and a presenter protein (e.g., FKBP) of greater than 1 μM,preferably greater than 5 μM and more preferably greater than 10 μM. Insome embodiments, target proteins which do not naturally associate witha presenter protein are those which are other than calcineurin or mTOR.

The selection of suitable target proteins for the disclosed assays maydepend on a presenter protein. For example, target proteins that havelow affinity for a cyclophilin may have high affinity for an FKBP andwould not be used together with the latter. Target proteins can benaturally occurring, e.g., wild type. In some embodiments, a targetprotein can vary from a wild type protein but still retain biologicalfunction, e.g., as an allelic variant, a splice mutant or a biologicallyactive fragment. In certain embodiments, a target protein is selectedfrom the group consisting of K-Ras, N-Ras, H-Ras, c-Raf, c-Myc, N-Myc,L-Myc, beta-catenin, MITF, Hif-1 alpha, Hif-2 alpha, PKN3, Bcl6, E2F1,AAC-11, PCSK 9, EIF4E, PLD1, PLD2, AAC-11, Frizzled7, c-Src, Fak, RaLP,Pyk2, NF-kappaB, MLL-1, Myb, Ezh2, Stat3, Stat5, c-Fos, C-Jun, RhoGD12,AMPK, EGFR CTLA4, Rab25, Rab11, AR(coact), ER(coact), GCGC (coact),Adiponectin R2, GPR 81, and IMPDH2. In some embodiments, a targetprotein is selected from the group consisting of IL-4R, IL-13R, IL-IR,IL2-R, IL-6R, IL-22R TNF-R, TLR4, Nrlp3, TRKB, STEP, OTR, Tau, andNav1.7

In some embodiments of a provided method, a Test Compound is contactedwith a presenter protein under conditions suitable for the formation ofa binary complex. In some embodiments, a Test Compound comprises aPresenter Interacting Moiety that is known to bind to the presenterprotein being utilized in the assay. In such cases, conditions forforming such binary complexes are known to the skilled person. Suitableconditions for Test Compounds comprising a rapamycin PresenterInteracting Moiety can be found in Bierer, B. E., Mattila, P. S.,Standaert, R. F., Herzenberg, L. A., Burakoff, S. J., Crabtree, G. &Schreiber, S. L. Proc. Natl. Acad. Sci. USA 87, 9231-9235 (1990).;suitable conditions for Test Compounds comprising an FK506 PresenterInteracting Moiety can be found in Harding M W, Galat A, Uehling D E,Schreiber S. L. Nature; 341(6244):758-60 (1989); suitable conditions forTest Compounds comprising an cyclosporine Presenter Interacting Moietycan be found in Handschumacher R E, Harding M W, Rice J, Drugge R J,Speicher D W Science; 226(4674):544-7 (1984); and suitable conditionsfor Test Compounds comprising a sanglifehrin Presenter InteractingMoiety can be found in WO2010/034243. The entire teachings of thesereferences are incorporated herein by reference. Suitable conditions forcompounds comprising a Presenter Interacting Moiety which variesstructurally from those listed above can be determined through routinevariation of binding conditions for known Presentation Ligands.

Once a binary complex has formed, it is contacted with a target proteinor target proteins. Target proteins can be in a purified form and simplyadded to an assay. In some embodiments, a binary complex is contactedwith cell lysates containing target proteins of interest. In someembodiments, cell lysates are of neuronal origin, cardiac origin, ormyeloid origin, or are lysates of cancer cell lines or lymphoid tissue.In some embodiments, cell lysates are from microorganisms. In someembodiments, cell lysates are from primary cells. In some embodiments,cell lysates are derived from any cell having an immunophilin orcyclophilin. The contacting is conducted under conditions known to formternary complexes between presenter protein, Presentation Ligands andtarget proteins, e.g., as taught in Liu J, Farmer J D Jr, Lane W S,Friedman J, Weissman I, Schreiber S L. Cell. 1991 Aug. 23; 66(4):807-15;Vogel K et al Advances in Protein Chemistry Volume 56, 2001, Pages253-291.

After contacting a binary complex with a target protein, the mixture isassessed to determine the affinity of the binary complex for the targetprotein is increased relative to either the (i) affinity of a compoundfor a target protein in the absence of the binary complex; or (ii) theaffinity of a presenter protein for a target protein in the absence ofthe binary complex. Where a target protein is known to not substantiallybind to a presenter protein or Test Compound in the absence of thebinary complex, identification of a ternary complex is indicative of anincrease in affinity. Binding can be determined by any suitable means,as discussed above. A preferred means is by tandem mass spectrometry.Where there is detectable binding between a target protein and a TestCompound or presenter protein, affinities of resulting complexes aremeasured, as discussed above.

One common way of carrying out a provided assay is by affinitypurification of protein complexes followed by mass spectrometricanalysis. As described below, this approach can be deployed to evaluatelibraries of small molecule ligands for their ability to enable novelprotein-protein interactions. In brief, “the bait”, an epitope taggedversion of a presenter protein e.g. the human FKBP or cyclophilinprotein can be generated and used to “fish” for ligand-mediatedprotein-protein events. The bound proteins can then be identified byproteolytic digestion, analytical analysis by liquidchromatography-tandem mass spectrometry and computational searchalgorithms to identify the peptides and their constituent proteins (seethe FIGURE).

It will be appreciated that a variety of methods can be used to analyzethe influence of small molecules on protein-protein interactions. Forexample, one basic procedure for generating an affinity column is toappend a cDNA of interest (human immunophilins/FKBPs or cyclophilins)with an epitope tag consisting of a polypeptide added to the N- orC-terminus of the cDNA open reading frame. This modified cDNA in anappropriate expression vector is then introduced into a suitable hoste.g. a mammalian cell line or bacterial strain, at which time the cDNAis transcribed and translated by the cellular machinery to generate theepitope tagged recombinant protein. The cells can be lysed underappropriate non-denaturing conditions by a number of methods includingsonication, French press, bead milling, treatment with lytic enzymes(e.g., lysozyme) or use of a commercially available cell lysis reagentsuch as the FastBreak™ Cell Lysis Reagent (Promega Inc.). Affinitycapture purification is then performed following a procedure that isbased on the affinity properties of the tag (Nilsson, J. et al ProteinExpr Purif. 11(1):1-16. (1997)). Many different affinity tags have beendeveloped to simplify protein purification including poly-histidine,DDK, Glutathione-S-Transferase-glutathione (GST-GSH), Hemagluttin (HA),and others (Terpe, K. Appl Microbiol Biotechnol., Appl MicrobiolBiotechnol., 60(5):523-33 (2003). In brief, this affinity tagged baitprotein is first purified from its production source (bacterial cells,mammalian cells, etc.) and then added to cell lysate generated from anorganisms or source of interest (eg. Human cell lines, etc) resulting ina mixture to which compounds can either be added or not. Afterincubation for sufficient time as to allow for protein complex formation(ie. Presenter-compound-target(s)), the bait protein and associatedproteins are purified using the strategy appropriate for the epitope tagof the bait (presenter) protein. These purified proteins are thendigested into peptides (either on column or after elution usingappropriate methods) using a suitable proteinase (Trypsin,Endoproteinase Lys-C, etc.) and this resulting peptide mixture is thematerial that will be analyzed using mass spectrometry methodologies(tandem mass spectrometry, MS3 methods, etc.). Below is described oneexample of how such a process could be used for identification of targetproteins for a bait (presenter) protein of interest wherein the baitprotein is fused at its amino terminus to GS, however, appropriatemodifications to this described methodology would be adopted if otherpurification tags are utilized and alterations to the purification,elution, and mass spectrometry-based analysis may also be made as theneeds of the assay are altered as necessary.

Protein purification using the GST-GSH system relies on the highaffinity of GST for immobilized GSH which allows selective proteinpurification (Hutchens, T. W., and Yip, T. T., J Inorg Biochem. 1;42(2):105-18 (1990). Hutchens, T. W., and Yip T. T., J Chromatogr.500:531-42 (1990)). The GSH is covalently coupled to Sepharose 4B via10-carbon spacer arm. Sepharose is a crosslinked, beaded-form ofagarose. GST can be appended to the amino or carboxyl terminal of aprotein via recombinant DNA technology thus enabling affinitypurification of the tagged protein based on GSH-GST interaction.Appropriate washing steps will reduce non-specific binding events to theGSH-sepharose matrix.

An affinity tagged recombinant immunophilin/FKBP or cyclophilin capturedon the solid phase support will then be probed with non-denatured cellextracts either untreated or treated (either pre- or post lysis) withsmall molecule ligands that comprise the libraries for evaluation.Ligands may be evaluated as single agents or as groups of compounds.Cell lines for evaluation will preferentially be human but may alsoinclude any cell lines that express immunophilins. In some embodiments,cell lines may be both immortalized and represent the National CancerInstitute 60 panel screening set (NCI-60):

Cell Line Name Panel Name Cell Line Name Panel Name CCRF-CEM LeukemiaLOX IMVI Melanoma HL-60(TB) Leukemia MALME-3M Melanoma K-562 LeukemiaM14 Melanoma MOLT-4 Leukemia MDA-MB-435 Melanoma RPMI-8226 LeukemiaSK-MEL-2 Melanoma SR Leukemia SK-MEL-28 Melanoma A549/ATCC Non-SmallCell SK-MEL-5 Melanoma Lung EKVX Non-Small Cell UACC-257 Melanoma LungHOP-62 Non-Small Cell UACC-62 Melanoma Lung HOP-92 Non-Small CellIGR-OV1 Ovarian Lung NCI-H226 Non-Small Cell OVCAR-3 Ovarian LungNCI-H23 Non-Small Cell OVCAR-4 Ovarian Lung NCI-H322M Non-Small CellOVCAR-5 Ovarian Lung NCI-H460 Non-Small Cell OVCAR-8 Ovarian LungNCI-H522 Non-Small Cell NCI/ADR-RES Ovarian Lung COLO 205 Colon SK-OV-3Ovarian HCC-2998 Colon 786-0 Renal HCT-116 Colon A498 Renal HCT-15 ColonACHN Renal HT29 Colon CAKI-1 Renal KM12 Colon RXF 393 Renal SW-620 ColonSN12C Renal SF-268 CNS TK-10 Renal SF-295 CNS UO-31 Renal SF-539 CNSMCF7 Breast SNB-19 CNS MDA-MB-231/ATCC Breast SNB-75 CNS MDA-MB-468Breast U251 CNS HS 578T Breast PC-3 Prostate MDA-N Breast DU-145Prostate BT-549 Breast T-47D Breast

In some embodiments, immortalized cell lines are selected from thefollowing:

Cell Line Panel Cell Line Name Panel Name Name Name LXFL 529 Non-SmallCell RPMI-7951 Melanoma Lung DMS 114 Small Cell M19-MEL Melanoma LungSHP-77 Small Cell RXF-631 Renal Lung DLD-1 Colon SN12K1 Renal KM20L2Colon P388 Leukemia SNB-78 CNS P388/ADR Leukemia XF 498 CNS

Profiling could also involve additional immortalized cell lines oflymphoid, myeloid, cardiac, neurona, pancreatic β muscle, fat cells andothers cell types representative of different human organ and tissuetypes. Experiments may also use lysates from primary sources includingcells of the immune system e.g. T, B, dendritic, nervous system e.g.cortical, hippocampal, and others. Appropriate washing steps will beused to reduce non-specific binding events followed by elution of theprotein complexes from the affinity column using increasingconcentrations of GSH, an affinity based-competitor of the GST epitopetag on the bait protein. The eluted material will then be evaluated viamass spectrometry.

Sample analysis requires tryptic digestion of the eluted material fromthe affinity column and subsequent analytical characterization usingliquid chromatography-tandem mass spectrometry (LC-MS/MS) which is ananalytical chemistry technique that combines the physical separationcapabilities of liquid chromatography (or HPLC) with the mass analysiscapabilities of mass spectrometry. LC-MS/MS has very high sensitivityand selectivity and it is used for determining masses of particles, fordetermining the elemental composition of a sample or molecule, and forelucidating the chemical constituents of molecules, including peptidesequences which can then be subsequently annotated into protein sequencevia the use of database searches and highly refined algorithms such asSequest, Mascot, OMSSA or others. The methods outlined above are meantto be representative but not exclusive technological solutions toaffinity purification and protein identification. For recent reviews onthe topic please see Liang, S. et al Current Proteomics 6:25-31 (2009).

Steps for carrying out a provided assay can be repeated with one or moreTest Compounds, for example, with a library of Test Compounds. An assaywith multiple Test Compounds can be carried out simultaneously orconcurrently; or can be carried out simultaneously with some TestCompounds and then concurrently with others.

Test Compounds which increase the affinity of a binary complex for atarget protein relative to the affinity of the Test Compound alone or apresenter protein alone can be selected for more advanced biologicaltesting. Test Compounds selected for more advanced testing andPresentation Ligands are collectively referred to herein as “LigandCompounds”. Suitable selection criteria for more advanced testinginclude a two-fold, five-fold, ten-fold or twenty-fold increase in theaffinity of a binary complex for a target protein relative to theaffinity of a Test Compound alone or a presenter protein alone. Forexample, selected Test Compounds can be tested using in vitro assays toassess whether they increase or decrease the activity of a targetprotein and to assess the magnitude of the increase or decrease.Suitable in vitro assays will, of course, depend on a target protein andwill likely require the presence of a presenter protein. In someembodiments, selected Test Compounds are tested in assays designed toassess efficacy against a disease condition or symptom(s) of a diseasecondition that is mediated by a target protein. For example, TestCompounds selected for their ability to modulate the activity of atarget protein implicated in the development and maintenance of cancercan be assessed for their ability to inhibit the growth cancer celllines. Suitable assays again will depend upon a target protein and arewell known to the skilled artisan. Exemplary assays are provided in thefollowing paragraphs.

Transcription Factors Bel-6

-   -   Bel-6 DNA binding and transcriptional activation    -   The BCL6 proto-oncogene suppresses p53 expression in        germinal-centre B cells. Phan,    -   RT, Dalla-Favera R. (2004). Nature 432(7017):635-9.        c-FOS    -   c-FOS/c-JUN interaction and luciferase reporter assay    -   Kinetic studies of Fos-Jun.DNA complex formation: DNA binding        prior to dimerization.    -   Kohler J J, Schepartz A. Biochemistry 40(1):130-42 (2001).    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA BioSciences Corporation 6951 Executive Way        Frederick, MD 21703 USA        c-JUN    -   FOS/c-JUN interaction and luciferase reporter assay    -   Kinetic studies of Fos-Jun. DNA complex formation: DNA binding        prior to dimerization.    -   Kohler J J, Schepartz A. Biochemistry 40(1):130-42 (2001).    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Scott Pattison. SA BioSciences        Corporation 6951 Executive Way Frederick, MD 21703 USA

E2F1

-   -   E2F1 DNA binding and transcriptional repression    -   Transcriptional repression of the prosurvival endoplasmic        reticulum chaperone    -   GRP78/BIP by E2F1. Racek T, Buhlmann S, Rüst F, Knoll S, Alla V,        Pützer BM. J Biol Chem. 283(49):34305-14 (2008). Epub 2008 Oct.        7.

Hif-1a

-   -   Hif-1a Reporter Assay    -   Technical Note    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA    -   Chemiluminescent Hif-1a transcription factor assay (HRP-based)    -   Identification of Small Molecule Inhibitors of Hypoxia-Inducible        Factor 1    -   Transcriptional Activation Pathway. Rapisarda, A., Uranchimeg,        B., Scudiero, D. A., Selby, M., Sausville, E. A., Shoemaker, R.        H., and Melillo, G. Canc. Res., 62:4316-4324 (2002).    -   Hif-1a redistribution assay    -   http://www.thermoscientific.jp/cellomics/redistribution/docs/HIF-1        alpha-U2OS.pdf    -   Flavonoids induce HIF-1 alpha but impair its nuclear        accumulation and activity.    -   Triantafyllou A, Mylonis I, Simos G, Bonanou S, Tsakalof A. Free        Radic Biol Med. 44(4):657-70 (2008). Epub 2007 Nov. 7.    -   Hif-1 ELISA    -   http://www.activemotif.com/catalog/204/transam-hif-1.html    -   Anti-angiogenic effects of SN38 (active metabolite of        irinotecan): inhibition of hypoxia-inducible factor 1 alpha        (HIF-1a)/vascular endothelial growth factor (VEGF) expression of        glioma and growth of endothelial cells. Hiroshi Kamiyama, Shingo        Takano, Koji Tsuboi and Akira Matsumura. Journal of Cancer        Research and Clinical Oncology Volume 131, Number 4, 205-213        (2005)

Hif-2A

-   -   Hif 2a and DNA binding    -   Cooperative interaction of hypoxia-inducible factor-2 alpha        (HIF-2 alpha) and Ets-1 in the transcriptional activation of        vascular endothelial growth factor receptor-2 (Flk-1). Elvert G,        Kappel A, Heidenreich R, Englmeier U, Lanz S, Acker T, Rauter M,        Plate K, Sieweke M, Breier G. J Biol Chem. 2003 Feb. 28;        278(9):7520-30 (2002). Epub 2002 Dec. 2.

MITF

-   -   MITF DNA binding and transcriptional activation    -   Microphthalmia-associated transcription factor is a critical        transcriptional regulator of melanoma inhibitor of apoptosis in        melanomas. Dynek J N, Chan S M, Liu J, Zha J, Fairbrother W J,        Vucic D. Cancer Res.; 68(9):3124-32 (2008).    -   Construction of Protein Chip to Detect Binding of Mitf Protein        (Microphthalmia Transcription Factor) and E-box DNA.    -   Sang-Hee Yang, Jung-Sun Han, Seung-Hak Baek, Eun-Young Kwak, Hae        Jong Kim, Jeong-Hyun Shin, Bong-Hyun Chung and Eun-Ki Kim.        Applied Biochemistry and Biotechnology Volume 151, Numbers 2-3,        273-282 (2008).

ChIP MITF

-   -   STAT3 and MITF cooperatively induce cellular transformation        through upregulation of c-fos expression. Akiko Joo, Hiroyuki        Aburatani, Eiichi Morii, Hideo Iba and Akihiko Yoshimur Oncogene        23, 726-734 (2004).

Myc Family

-   -   myc-responsive luciferase reporter    -   Technical Note    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds.Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA    -   FRET-based binding of tagged myc    -   AlphaLISA Immunoassay Platform—the “No-Wash” High-Throughput        Alternative to ELISA. Bielefeld-Sevigny, M. Assay Drug Dev        Technol 7, 90-92 (2009).    -   ELISA w/dsDNA for pulldown    -   TEAL7 Inhibition of c-Myc Activity in Alternative Lengthening of        Telomeres Regulates hTERT Expression. Kyle Lafferty-Whyte, Alan        Bilsland, Stacey F Hoare, Sharon Burns, Nadia Zaffaroni, Claire        J Cairney, and William Nicol Keith. Neoplasia 12(5): 405-414        (2010).

MLL-1

-   -   MLL-1 and transcriptional regulatory activity    -   Cooperativity in transcription factor binding to the coactivator        CREB-binding protein (CBP). The mixed lineage leukemia protein        (MLL) activation domain binds to an allosteric site on the KIX        domain. Goto N K, Zor T, Martinez-Yamout M, Dyson H J,        Wright P. E. J Biol Chem.; 277(45):43168-74 (2002). Epub 2002        Aug. 29.    -   Domains with transcriptional regulatory activity within the ALL1        and AF4 proteins involved in acute leukemia. R Prasad, T Yano, C        Sorio, T Nakamura, R Rallapalli, Y Gu, D Leshkowitz, C M Croce,        and E Canaani. Proc Natl Acad Sci USA. 92(26): 12160-12164        (1995).

Myb

-   -   Protein-protein Interaction and transcriptional regulation    -   Detection of proteins that bind to the leucine zipper motif of        c-Myb. Favier D, Gonda T J. Oncogene. January; 9(1):305-11        (1994).    -   Pim-1 kinase and p100 cooperate to enhance c-Myb activity.        Leverson J D, Koskinen P J, Orrico F C, Rainio E M, Jalkanen K        J, Dash A B, Eisenman R N, Ness S A. (1998). Mol Cell;        2(4):417-25.

NF-κB

-   -   DNA Binding and complex formation    -   Competition between TRAF2 and TRAF6 regulates NF-kappaB        activation in human B lymphocytes. Zhang W, Zhang X, Wu X L, He        L S, Zeng X F, Crammer A C, Lipsky P E. Chin Med Sci J. (1):1-12        (2010).    -   Visualization of AP-1 NF-kappaB ternary complexes in living        cells by using a BiFC-based FRET. Shyu Y J, Suarez C D, Hu C D.        Proc Natl Acad Sci USA. 2008 Jan. 8; 105(1):151-6 (2008). Epub        2008 Jan. 2.    -   Technical Note    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of and Scott Pattison. SA Biosciences        Corporation 6951 Executive Way Frederick, MD 21703 USA

Stat3

-   -   DNA Binding and complex formation    -   A novel small-molecule disrupts Stat3 SH2 domain-phosphotyrosine        interactions and Stat3-dependent tumor processes. Zhang X, Yue        P, Fletcher S, Zhao W, Gunning P T, Turkson J. Biochem        Pharmacol.; 79(10):1398-409 (2010). Epub 2010 Jan. 11.    -   Technical Note    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA

Stat5

-   -   DNA Binding and complex formation Mammalian protein-protein        interaction trap (MAPPIT) analysis of STAT5, CIS, and SOCS2        interactions with the growth hormone receptor. Uyttendaele I,        Lemmens I, Verhee A, De Smet A S, Vandekerckhove J, Lavens D,        Peelman F, Tavernier J. Mol Endocrinol. (11):2821-31 (2007).        Epub 2007 Jul. 31.    -   Technical Note    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA

Wnt/Beta-Catenin Beta-Catenin Redistribution Assay—Nuclear Translocationof EGFP-Beta-Catenin

-   -   Cadherin-bound beta-catenin feeds into the Wnt pathway upon        adherens junctions dissociation: evidence for an intersection        between beta-catenin pools.    -   Kam Y, Quaranta V. PLoS One; 4(2):e4580 (2009). Epub 2009 Feb.        24.

Wnt/Beta-Catenin Signaling Via Fluorescent Reporter

-   -   Fluorescence-based functional assay for Wnt/beta-catenin        signaling activity.    -   Zhou L, An N, Jiang W, Haydon R, Cheng H, Zhou Q, Breyer B, Feng        T, He T C. Biotechniques 33(5):1126-8 (2002)

Nuclear Hormone Receptors Androgen Receptor

-   -   AR and Co-activators    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA    -   Functional screening of FxxLF-like peptide motifs identifies        SMARCD1/BAF60a as an androgen receptor cofactor that modulates        TMPRSS2 expression. van de Wijngaart D J, Dubbink H J, Molier M,        de Vos C, Trapman J, Jenster G. Mol Endocrinol. (11):1776-86        (2009). Epub 2009 Sep. 17.

Estrogen Receptor ER and Co-Activators

-   -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of nd Scott Pattison. SA Biosciences        Corporation 6951 Executive Way Frederick, MD 21703 USA    -   A set of time-resolved fluorescence resonance energy transfer        assays for the discovery of inhibitors of estrogen        receptor-coactivator binding. Gunther J R, Du Y, Rhoden E, Lewis        I, Revennaugh B, Moore T W, Kim S H, Dingledine R, Fu H,        Katzenellenbogen J A. J Biomol Screen. (2):181-93 (2009). Epub        2009 Feb. 4.

Signal Transducers/Adaptors Frizzled7

-   -   Frizzled7 protein-protein interactions with PSD-95    -   Direct interaction of Frizzled-1, -2, -4, and -7 with PDZ        domains of PSD-95. Hering H, & Sheng M, FEBS Lett.;        521(1-3):185-9 (2002).

Rab25

-   -   Rab25 protein-protein interaction with growth factor receptors    -   Emerging Role of RAB GTPases in Cancer and Human Disease.        Kwai W. Cheng, John P. Lahad, Joseph W. Gray, and Gordon B.        Mills. Cancer Res. 65; 2516 (2005).    -   Development of new tools to comprehensively analyze mammalian        Rabs in membrane traffic. Fukuda M. Histol Histopathol.        (11):1473-80 (2010).

RAS and Raf

-   -   Ras assay reagent—Raf1-RBD on agarose    -   Minimal Ras-binding domain of RAF-1 can be used as an activation        specific probe for Ras.de Rooij, J and Bos, J. L. Oncogene, 14:        623-5 (1997)    -   Ras GTPase Activation ELISA Kit—Raf1-RBD capture and        anti-RAS+HRP Imatinib blocks migration and invasion of        medulloblastoma cells by concurrently inhibiting activation of        platelet-derived growth factor receptor and transactivation of        epidermal growth factor receptor. Abouantoun T J, Macdonald T J.        Mol Cancer Ther. Nov. 1, (2010).    -   Raf1-RBD on beads    -   Protein kinase C (PKC) βII induces cell invasion through a        Ras/Mek-, PKC √Rac 1-dependent signaling pathway. Zhang, J.,        Anastasiadis, P. Z., Liu, Y., Thompson, E. A. and        Fields, A. P. J. Biol. Chem. 279, 22118-22123 (2004).    -   Ras-Raf co-precipitation    -   Critical Binding and Regulatory Interactions between Ras and Raf        Occur through a Small, Stable N-Terminal Domain of Raf and        Specific Ras Effector Residues. Chuang, E., Barnard, D.,        Hetfich, L. A., Zhang, X F., Avruch, J., and Marshall, M. S.        Mol. Cell. Biol., Vol. 14, No. 8: 5318-5325 (1994).    -   Rho GDP dissociation inhibitor 2 (RhoGDI2)    -   RhoGD12 and Rac-1 interaction    -   RhoGDI2 as a therapeutic target in cancer. Cho H J, Baek K E,        Yoo J. Expert Opin Ther Targets. 4(1):67-75 (2010).

PLD-1

-   -   PLD-1 Protein-Protein interaction with PKCa    -   Mechanisms of regulation of phospholipase D1 by protein kinase C        alpha.Hu T, Exton J H. J Biol Chem. 278(4):2348-55 (2003).

PLD-2

-   -   PLD-1 protein-protein interaction with PKCgamma-1    -   The direct interaction of phospholipase C-gamma 1 with        phospholipase D2 is important for epidermal growth factor        signaling. Jang I H, Lee S, Park J B, Kim J H, Lee C S, Hur E M,        Kim I S, Kim K T, Yagisawa H, Suh P G, Ryu S H. J Biol Chem.        278(20):18184-90 (2003). Epub 2003 Mar. 19.

Kinases

c-Src

-   -   c-Src protein-protein interaction    -   Focal Adhesion Kinase Overexpression Enhances Ras-dependent        Integrin Signaling to    -   ERK2/Mitogen-activated Protein Kinase through Interactions with        and Activation of c-Src. David D. Schlaepfer and Tony Hunter. J.        Biol. Chem. Vol. 272, 20: 13189-13195 (1997).

FAK

-   -   FAK protein-protein interaction    -   Focal Adhesion Kinase Overexpression Enhances Ras-dependent        Integrin Signaling to ERK2/Mitogen-activated Protein Kinase        through Interactions with and Activation of c-Src. David D.        Schlaepfer and Tony Hunter. J. Biol. Chem. Vol. 272, 20:        13189-13195 (1997).

P38

-   -   Millipore—p38 MAP Kinase Assay    -   Millipore's HCS231 p38 MAP Kinase Assay provides a complete        solution for identifying and quantifying the phosphorylation        state of endogenous cellular p38    -   www.millipore.com/catalogue/item/HCS231    -   Many others commercially available

PKN3

-   -   PKN3 catalytic activity    -   PKN3 is required for malignant prostate cell growth downstream        of activated PI 3-kinase.

Frauke Leenders,¹ Kristin Möpert,¹ Anett Schmiedeknecht,¹ AnsgarSantel,¹ Frank Czauderna,¹ Manuela Aleku,¹ Silke Penschuck,^(1*)SibylleDames,¹ Maria Sternberger,¹ Thomas Röhl,¹ Axel Wellmann, WolfgangArnold, Klaus Giese,¹ Jörg Kaufmann,¹ and Anke Klippel. EMBO J.23(16):3303-3313 (2004). Published online 2004 Jul. 29

-   -   Pyk2-proline-rich tyrosine kinase-2    -   PyK2 and PDK1 protein-protein Interaction Pyk2- and        Src-dependent tyrosine phosphorylation of PDK1 regulates focal        adhesions. Taniyama, Y., Weber, D. S., Rocic, P., Hilenski, L.,        Akers, M. L., Park, J., Hemmings, B. A., Alexander, R. W.,        Griendling, K. K. Mol. Cell. Biol. (22):8019-29 (2003).

RaLP

-   -   RalP protein-protein interaction    -   RaLP, a new member of the Src homology and collagen family,        regulates cell migration and tumor growth of metastatic        melanomas. Fagiani E, Giardina G, Luzi L, Cesaroni M, Quarto M,        Capra M, Germano G, Bono M, Capillo M, Pelicci P, Lanfrancone L.        Cancer Res.;67(7):3064-73 (2007).

ZAP-70

-   -   Zeta-chain-associated protein kinase 70    -   A Novel ZAP-70 Dependent FRET Based Biosensor Reveals Kinase        Activity at both the Immunological Synapse and the Antisynapse        Randriamampita C, Mouchacca P, Malissen B, Marguet D, Trautmann        A, et al. PLoS ONE 3(1): e1521 (2008).    -   ZAP70 Kinase Enzyme System www.promega.com/Catalogue #V8311    -   Many others commercially available

Kinases General:

-   -   Ambit platform: http://www.ambitbio.com/    -   Fluorescence lifetime after displacement of labeled        staurosporine:    -   http://jbx.sagepub.com/content/12/6/828.abstract    -   FRET of tagged kinase (b/w labeled antibody and        tracer)—Invitrogen LanthaScreen    -   http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Drug-Di        scovery/Target-and-Lead-Identification-and-Validation/KinaseBiology/Kinase-Activity-Assays/lanthascreentm-eu-kinase-binding-assay.html    -   Tritium labeled pyridinyl imidazole:    -   http://jbx.sagepub.com/content/4/3/129.abstract

Cytokine, GF, TNF, Toll Receptors IL-4R

-   -   Analysis of promoter regions regulating basal and        interleukin-4-inducible expression of the human CB1 receptor        gene in T lymphocytes. Börner C, Bedini A, Höllt V, Kraus J. Mol        Pharmacol. 73(3):1013-9 (2008). Epub 2007 Dec. 21

IL-13R

-   -   Elevated IL-13 Ralpha2 in intestinal epithelial cells from        ulcerative colitis or colorectal cancer initiates MAPK pathway.        Mandal D, Levine A D. Inflamm Bowel Dis., 16(5):753-64 (2010).

IL-1R

-   -   MyD88, IRAK1 and TRAF6 knockdown in human chondrocytes inhibits        interleukin-1-induced matrix metalloproteinase-13 gene        expression and promoter activity by impairing MAP kinase        activation. R. Ahmad, J. Sylvester, and M. Zafarullah, “Cellular        Signalling, vol. 19, no. 12, pp. 2549-2557 (2007).

IL-2R

-   -   Protein phosphatase 2A regulates interleukin-2 receptor complex        formation and JAK3/STAT5 activation. Ross J A, Cheng H, Nagy Z        S, Frost J A, Kirken R A. J Biol Chem; 285(6):3582-91 (2010).        Epub 2009 Nov. 18.

IL-6R

-   -   The full-length leptin receptor has signaling capabilities of        interleukin 6-type cytokine receptors. Baumann H, Morella K K,        White D W, Dembski M, Bailon P S, Kim H, Lai C F, Tartaglia L A.        Proc Natl Acad Sci USA. 93(16):8374-8 (1996).    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of d Scott Pattison. SA Biosciences        Corporation 6951 Executive Way Frederick, MD 21703 USA

IGF-1R

-   -   A novel, potent, and selective insulin-like growth factor-I        receptor kinase inhibitor blocks insulin-like growth factor-I        receptor signaling in vitro and inhibits insulin-like growth,        Gibson N W, Pachter J A. Mol Cancer Ther. 6 (8):2158-67 (2007).        Epub 2007 Aug. 1.

TNF-R

-   -   NF-kB activation: TECHNICAL NOTE    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA

TLR4

-   -   LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll        adapters TRAM and TRIF. Fitzgerald K A, Rowe D C, Barnes B J,        Caffrey D R, Visintin A, Latz E, Monks B, Pitha P M, Golenbock        D T. J Exp Med.;198(7):1043-55 (2003). Epub 2003 Sep. 29.    -   NF-κB activation: TECHNICAL NOTE    -   Cignal™ Reporter Assay Kit: A High Performance Tool for        Assessing the Functions of Genes, Biologics and Small Molecule        Compounds. Vikram Devgan, Abigail Harris, Qiong Jiang, and Scott        Pattison. SA Biosciences Corporation 6951 Executive Way        Frederick, MD 21703 USA.

Miscellaneous AAC-11

-   -   AAC-11 functional readout    -   The antiapoptotic protein AAC-11 interacts with and regulates        Acinus-mediated DNA fragmentation. Rigou P, Piddubnyak V, Faye        A, Rain J C, Michel L, Calvo F, Poyet J L.    -   EMBO J. 28(11):1576-88 (2009). Epub 2009 Apr. 23.

CTLA4

-   -   CTLA4-targeting intracellular domain protein-protein        interactions.    -   Targeting metastatic melanoma (2008) Poust J. Am J Health Syst        Pharm. 2008 Dec. 15; 65 (24 Suppl 9):S9-S15.    -   Inhibition of CTLA-4 function by the regulatory subunit of        serine/threonine phosphatase 2A. Baroja M L, Vijayakrishnan L,        Bettelli E, Darlington P J, Chau T A, Ling V, Collins M, Carreno        B M, Madrenas J, Kuchroo V K. J Immunol.; 168(10):5070-8 (2002).

EIF4E EIF4E Activity and Protein-Protein Interaction

-   -   Regulation of eukaryotic initiation factor 4E (eIF4E)        phosphorylation by mitogen-activated protein kinase occurs        through modulation of Mnk1-eIF4G interaction.    -   Shveygert M, Kaiser C, Bradrick S S, Gromeier M. Mol Cell Biol.        (21):5160-7. Epub 2010 Sep. 7 (2010).

Ezh2-Histone Demethylase

-   -   Coordinated activities of wild-type plus mutant EZH2 drive        tumor-associated hypertrimethylation of lysine 27 on histone H3        (H3K27) in human B-cell lymphomas.    -   Sneeringer C J, Scott M P, Kuntz K W, Knutson S K, Pollock R M,        Richon V M, Copeland R A. Proc Natl Acad Sci. (2010) Nov. 15.        [Epub ahead of print]

IMPDH2

-   -   Inhibition by mycopheolic acid Effect of mycophenolate        acyl-glucuronide on human recombinant type 2 inosine        monophosphate dehydrogenase. Gensburger O, Picard N, Marquet P.        Clin Chem.;55(5):986-93 (2009). Epub 2009 Mar. 19.

Nav1.7

-   -   Measure voltage-gated channel opening    -   Characterization of voltage-gated sodium-channel blockers by        electrical stimulation and fluorescence detection of membrane        potential. Chien-Jung Huang, Alec Harootunian, Michael P Maher,        Catherine Quan, Christopher D Raj, Ken McCormack, Randal Numann,        Paul A Negulescu, & Jesns E Gonzilez, Nature Biotechnology 24,        439-446 (2006).

NRLP3

-   -   Manji G A, Wang L, Geddes B J, et al. “PYPAF1, a        PYRIN-containing Apaf1-like protein that assembles with ASC and        regulates activation of NF-kappa B.”. J. Biol. Chem. 277 (13):        11570-5 (2002)

PTP-1B

-   -   Docking simulations and in vitro assay unveil potent inhibitory        action of papaverine against protein tyrosine phosphatase 1B.        Bustanji Y, Taha M O, Al-Masri I M, Mohammad M K. Biol Pharm        Bull.;32(4):640-5 (2009).    -   Synthesis, in vitro and computational studies of protein        tyrosine phosphatase 1B inhibition of a small library of        2-arylsulfonylaminobenzothiazoles with antihyperglycemic        activity. Navarrete-Vazquez G, Paoli P, León-Rivera I,        Villalobos-Molina R, Medina-Franco J L, Ortiz-Andrade R,        Estrada-Soto S, Camici G, Diaz-Coutiño D, Gallardo-Ortiz I,        Martinez-Mayorga K, Moreno-Diaz H. Bioorg Med Chem.        17(9):3332-41 (2009). Epub 2009 Mar. 26.    -   PTP1B Assay Kit, Colorimetric—EMD4 Biosciences | EMD Chemicals        USA A colorimetric assay kit to measure PTP1B activity.

In many instances, drug candidates are developed having good efficacyagainst a desired target protein, only to fail during their drugdevelopment when it is discovered that they also have an undesiredactivity against a second target protein, leading to unacceptable sideeffects. For ease of reference, these drug candidates are referred to as“modulator compounds”. In some embodiments, the present inventionprovides methods of developing a drug candidate with modifiedselectivity towards a first target protein relative to a second targetprotein from a modulator compound that modulates the activity of boththe first and second target proteins. The methods disclosed hereinprovide, among other things, methods for modifying the structure ofmodulator compounds to develop new drug candidates having increasedselectivity for a desired target and decreased activity towards aundesired target. In some embodiments, a method utilizes a LigandCompound complexed with a presenter protein to a form binary compoundwith enhanced binding affinity towards a desired target protein relativeto an undesired target protein.

As discussed above, a Ligand Compound comprises a Presenter InteractingMoiety (a Conserved Region) and a Target Interacting Moiety (VariableRegion). A Presenter Interacting Moiety binds to a presenter protein toform a binary complex; and a Target Interacting Moiety enhances theaffinity of the resulting binary complex for a desired target proteinrelative to the affinity of a presenter protein for a desired targetprotein in the absence of the binary complex. In some embodiments, aTarget Interacting Moiety is selected so that it does not substantiallybind to an undesired target protein, e.g., binds to an undesired targetprotein with a K_(d) greater than 1 μM, preferably with a K_(d) greaterthan 10 μM. In some embodiments, a Modulator Compound is covalentlyassociated (bonded) to a Target Interacting Moiety of a Ligand Compoundto form a modified Ligand Compound. “Covalently associating” a ModulatorCompound to a Target Interacting Moiety of a Ligand Compound means thatthere is a covalent bond between a Target Interacting Moiety and aModulator Compound. In some embodiments, a Modulator Compound isconnected to a Target Interacting Moiety by a linker group. In someembodiments, a Modulator Compound is incorporated within a TargetInteracting Moiety as part of the macrocyclic ring itself. In someembodiments, a Modulator Compound is covalently bonded to two distinctring atoms in a Target Interacting Moiety and is the only group whichintervenes between these two ring atoms.

In some embodiments, after formation of a modified Ligand Compound, apresenter protein and a modified Ligand Compound are combined underconditions suitable for formation of a binary complex, referred toherein as a “modified binary complex”. Because a Modulator Compound isbonded to a Target Interacting Moiety, a Presenter Interacting Moietyremains free to bind with a presenter protein. As such, conditionsdescribed above for forming a binary complex with a Test Compound aresuitable for use in forming a modified binary complex with a modifiedLigand Compound. Because a binary complex of a Ligand Compound and apresenter protein shows selectivity towards a first target proteinrelative to a second target protein, associating the Modulator Compoundwith a binary complex will similarly impart a Modulator Compound withselectivity towards a desired first target. In certain embodiments,interacting sites form contacts between the target protein and themodulator compound, and the target protein and the Target InteractingMoiety. In certain embodiments, interacting sites form contacts betweenthe target protein and the modulator compound, the target protein andthe Target Interacting Moiety, and the target protein and the presenterprotein.

The activity of a first target protein is then measured in the presenceof a modified binary complex and in the presence of a modulator compound(alone, e.g., in the absence of a modified binary complex); and theactivity of a second protein is then measured in the presence of amodified binary complex and in the presence of a modulator compound(alone, e.g., in the absence of a modified binary complex). Methods formeasuring the activity of a second target protein are as described abovefor assays utilizing a Test Compound. The activity of first and secondtarget proteins in the presence of a modified binary complex and theactivity of first and second target proteins in the presence of amodulator compound (alone, e.g., without the modified binary complex)are compared to assess whether a modified ligand compound has alteredselectivity towards a first target protein relative to a second targetprotein compared with a modulator compound. Modified binary complexesthat show increased selectivity for a desired target protein can then beselected and tested in more advanced biological assays, as describedabove for Test Compounds.

The steps of the method can be repeated with additional modified binarycomplexes or by preparing additional modified Ligand Compounds andtesting them according to the steps just described. The activity of amodified binary complex and its selectivity for a desired target proteinrelative to an undesired target protein can be influenced by how aModulator Compound is associated with a Ligand Compound, e.g., where aModulator Compound is attached, whether it is directly bonded to theTarget Interacting Moiety of the Ligand Compound, incorporated into aTarget Interacting Moiety of a Ligand Compound, or whether it isconnected to a Target Interacting Moiety of a Ligand Compound through alinker group and the positioning of the attachment. Where a linker ispresent, the length, size and nature of the linker can also influenceactivity and selectivity. As such, it is desirable to repeat the assaywith multiple modified binary complexes, e.g., wherein the point ofassociation, the means of association and, where present, the length,size and nature of the linker are varied. Alternatively, or in additionto, the structure of a modulator compound and/or a Target InteractingMoiety of a Ligand Compound can be varied. Where a plurality of modifiedbinary complexes are tested, e.g., as in a library, compounds can betested simultaneously, concurrently or some simultaneously and othersconcurrently.

The relevant teachings of the publications discussed herein areincorporated herein by reference.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXEMPLIFICATION

Sequence validated cDNAs encoding the collection of either humanimmunophilins/FKBPS (FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23,FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60 and FKBP65) orcyclophilins (Cyp-A, PPIL1, PPIL3, USA-Cyp, Cyp-F, Cyp-B, Cyp-C, Cyp29,Cyp33, Cyp40, SDCCAG10, Cyp57, Cyp60, HAL539, Cyp88, NK-Cyp and RanBP)can be acquired from commercial sources (Origen, Inc.). Standardmolecular biology techniques can be utilized to clone the cDNA into anappropriate vector for expression in bacteria. One such vector ispEX-N-GST (Origen) that will result in an open reading frame (ORF) withan amino terminal GST affinity tag. GST-tags are often used for affinitypurification of recombinant proteins expressed in Escherichia coli andother prokaryotic expression systems. The cloning vector pEX-N-GST alsocontains an isopropyl-β-D-thio-galactoside (IPTG) regulated promoter toinduce expression of the cloned ORF. If desired, an ORF containing acarboxy terminal GST affinity tag can be generated using analternatively designed vector. Additional cloning vectors could also bechosen that will append the amino or carboxy terminus of the ORF with analternative affinity tag e.g poly-histidine, Flag, DDK, and others. Thevector containing the affinity tagged ORF (FKBP or cyclophilin) can betransformed into an appropriate expression strain such as E. coliBL21/DE3 using standard methods. Bacterial growth and where appropriateIPTG-induction is performed. Bacterial cells are harvested viacentrifugation and the resulting cell pellet lysed either by physicalmeans or by means of detergents and enzymes such as lysozyme followed bycentrifugation to generate a cleared cell lysate. The GST-taggedproteins can be purified from the cleared bacterial extract bychromatography over a glutathione (GSH)-agarose column (GE HealthCare)or via use of GSH magnetic resins (Pierce) or other GSH affinity capturemethods. Each method will utilize kit recommended buffers andconditions. However, each method consists of a general set of steps thatenable binding of the GST tagged protein to the GSH solid supportfollowed by appropriate washing steps to remove unwanted non-specificbinding proteins. These steps provide a prepared affinity capturedGST-tagged protein to be used for subsequent experimentation. Moreover,captured GST-tagged fusion protein can be eluted with PBS containing 30mM GSH to allow the purified tagged immunophilins and cyclophilins to beevaluated for both natural ligand binding (rapamycin, FK506 orcyclosporin) and peptidyl-prolyl isomerase activity as described in Liu,J., Albers, M. W., Chen, C. M., Schreiber, S. L., & Walsh, C. T. Proc.Natl. Acad. Sci. USA 87, 2304-2308 (1990); Liu, J., Farmer, J. D., Jr.,Lane, W. S., Friedman, J., Weissman, I., & Schreiber, S. L. Calcineurinis a common target of Cyclophilin-cyclosporin A and FKBP-FK506complexes. Cell 66, 807-815 (1991).

Mammalian cell extracts can be generated using standard commerciallyavailable kits such as CytoBuster Protein Extraction Reagent (Novagen,Inc). Briefly, lysates prepared from adherent cells required removal ofculture medium, two washes with phosphate buffered saline followed byaddition of the recommended amount of a non-denaturing mammalian celllysis reagent plus an appropriate protease inhibitor cocktail (seeproduct insert for lysis conditions); generally 5 min at 4° C. Celldebris are collected by centrifugation for 5 min at 16,000×g (4° C.).Supernatant is recovered for subsequent experimental analysis.Generating cell lysates from non-adherent cells requires a slow speedcentrifugation step (2,000×g) for 5 min to collect cells and subsequentmethod application as described above.

The mammalian cell extracts (0.4-4.0 mg protein/ml) can be pre-clearedby a 1 hr pre-incubation with GSH-sepahose using a 1:100 ration ofextract to sepharose resin) at 4° C. followed by a centrifugation step.This step will remove the endogenous glutathione binding proteinsincluding GST. The cleared extracts can then be mixed with theimmobilized tagged proteins and gently mixed for 1 hr in the absence orpresence of 10 μM of the individual Test Compounds. The GSH solidsupport can be washed via methods specific for each manufacturersproduct but generally entail intermittent centrifugation and washingsteps with a buffer such as PBS containing a mild detergent such as0.02% Nonidet P-40 or 0.2% Triton X-100. The washed GSH bound proteincomplexes can then be eluted via the use of 30-50 mM glutathione andprepared for mass spectrometry analysis.

Tandem mass spectrometer can “sequence” a peptide ion by first measuringthe mass of the peptide and then selectively isolating and gentlyfragmenting that peptide at peptide bonds followed by mass measurementof the fragment ions. The resulting tandem mass spectrum contains thesequence information for a single peptide. For mass spectrometryanalysis, the eluted protein complexes can be dialyzed to remove excessGSH. Proteins can then be TCA precipitated, trypsinized, andsubsequently purified using Empore C18 extraction media (3M), andanalyzed via LC-MS/MS; for example via use of a LTQ linear ion trap massspectrometer (ThermoFinnigan) using an 18 cm×125 μm (ID) C18 column anda 50 minute 8%-26% acetonitrile gradient. Spectra can then searchedusing highly refined algorithms such as Sequest, Mascot, OMSSA or othersagainst a target-decoy human tryptic peptide database to search andenable protein identification. Methods for each approach are asdescribed in Bauer, A and Kuster, B., Affinity purification-massspectrometry: Powerful tools for the characterization of proteincomplexes Eur. J. Biochem. 270, 570-578 (2003); Chang I. F., Massspectrometry-based proteomic analysis of the epitope-tag affinitypurified protein complexes in eukaryotes Current Proteomics 6, 6158-6166(2006); Sowa, M. E., Bennett, E. J., Gygi, S. P., and Harper J. W.Defining the Human Deubiquitinating Enzyme Interaction Landscape Cell;138(2): 389-403(2009).

If Test Compound dependent target proteins are identified, furtherstudies are performed to determine whether there is measurabledifference in affinity for the Test Compound and the identified targetin the absence versus the presence of the presenter protein. Standardmethods for this approach include calculation of the dissociationconstant (K_(d)). The K_(d) is a specific type of equilibrium constantthat measures the propensity of a larger object to separate (dissociate)reversibly into smaller components, as when a complex falls apart intoits component molecules.

There are multiple established technical methods to calculate Ks(reviewed in Stockwell, B. R. Nature; 432 (7019): 846-54 (2004)) andhave applicability to generate data to address questions as highlightedabove. Specific techniques such as Surface Plasmon Resonance (SPR) andFluorescence Polarization (FP) may represent preferred but not exclusivemethodologies for calculating K_(d). SPR measures the change inrefractivity of a metal surface when a compound binds to a protein thatis immobilized on that metal surface (Homola, J., Annal. Bioanal. Chem.;377: 528-539 (2003)) while FP measures the change in tumbling rate for acompound when it is bound to a protein using loss-of-polarization ofincident light (Burke, T. J., et. al. Comb. Chem. High ThroughputScreen; 6: 183-194 (2003)). Each of these technologies has provenamenable to calculating the K_(d) of protein-drug-protein complexes(Banaszynski, L. A., et. al. J. Am. Chem. Soc.; 127: 4715-4721 (2005).

Experiments are also performed to define whether formation of thepresenter protein-test compound interaction with the captured targetresults in modulation of the normal activity of the target. Thesemethods will be target specific and non-exhaustive examples have beenprovided in a proceeding section of this document.

1. A method of producing a complex, said method comprising contacting acompound and a presenter protein under suitable conditions, wherein thecomplex binds H-Ras or N-Ras and the compound has the formula:

wherein: the compound comprises between 18 and 30 ring atoms, whereineach of the ring atoms is selected from the group consisting of oxygen,nitrogen, carbon, sulfur, and phosphorus; each L is independently a bondor a bivalent substituted or unsubstituted portion of the compound; theTIS is a Target Interacting Site that contacts one or more correspondinginteracting sites on H-Ras or N-Ras; and the PIS is a PresenterInteracting Site that contacts one or more corresponding interactingsites on the presenter protein, and wherein the PIS comprises theformula selected from the formulas consisting of (A), (B) and (C):

wherein: J is hydrogen or (C1-C2) alkyl; K is (C1-C4)-straight orbranched alkyl, benzyl or cyclohexylmethyl; or wherein J and K may betaken together to form a 5-7 membered heterocyclic ring which maycontain an O, S, SO or SO₂ substituent therein; the stereochemistry atcarbon position 1 is R or S; X is selected from —NH—, —N(alkyl)-, —O—,—C(O)—, —CHOH—, —CH═, or —CH₂—; Y is selected from —C(O)NH—,—C(O)N(alkyl)-, —C(O)O—, —C(O)C(O)—, —C(O)CHOH—, —C(O)CH═, —C(O)CH₂—,and —S(O)₂; and

represents a single or a double bond; and the points of attachment tothe rest of the compound are through a terminus of each L; and

wherein: each X is independently selected from —NH—, —N(alkyl)-, —O—,—C(O)—, —CHOH—, or —CH₂—; and the points of attachment to the rest ofthe compound is by a covalent bond to a terminus of each L; and

wherein: X is selected from —NH—, —N(alkyl)-, —O—, —C(O)—, —CHOH—, or—CH₂—, R¹ is selected from (C1-C6)-alkyl, (C1-C6)-alkenyl,(C1-C6)-alkynyl, aryl, (C3-C7)-carbocyclyl, —(C1-C4 alkylene)-aryl, and—(C1-C4 alkylene)—(C3-C7) carbocyclyl; each R² is independently selectedfrom halo, —C≡N, C1-C4 alkyl, ═O, C3-C7 cycloalkyl, C1-C4 alkyl, —OH,—O—(C₁-C₄ alkyl), —SH, —S—(C₁-C₄ alkyl), —(C₁-C₄ alkyl)-N(R^(b))(R^(b)),—N(R^(b))(R^(b)), —O—(C₁-C₄ alkyl)-N(R^(b))(R^(b)), —(C₁-C₄alkyl)-O—(C₁-C₄ alkyl)-N(R^(b))(R^(b)), —C(O)—N(R^(b))(R^(b)), —(C₁-C₄alkyl)-C(O)—N(R^(b))(R^(b)), —O-(heteroaryl), —O- (heterocycle),—O-phenyl, -heteroaryl, -heterocycle, and -phenyl, wherein: each R^(b)is independently selected from hydrogen, and —C₁-C₄ alkyl; or two R^(b)are taken together with the nitrogen atom to which they are bound toform a 4- to 8-membered saturated heterocycle optionally comprising oneadditional heteroatom selected from N, S, S(═O), S(═O)₂, and O, anyalkyl substituent is optionally further substituted with one or more of—OH, —O—(C₁-C₄ alkyl), halo, —NH₂, —NH(C₁-C₄ alkyl), or —N(C₁-C₄alkyl)₂; and any carbon atom on a phenyl, cycloalkyl, heteroaryl orheterocycle substituent is optionally further substituted with one ormore of —(C₁-C₄ alkyl), —(C₁-C₄ fluoroalkyl), —OH, —O—(C₁-C₄ alkyl),—O—(C₁-C₄ fluoroalkyl), halo, —NH₂, —NH(C₁-C₄ alkyl), or —N(C₁-C₄alkyl)₂; m is 0, 1, 2 or 3; and the points of attachment to the rest ofthe compound is by a covalent bond to a terminus of each L; wherein eachL is independently selected from a bond and a linear chain of up to 10atoms, independently selected from carbon, nitrogen, oxygen, sulfur orphosphorous atoms, wherein each atom in the chain is optionallysubstituted with one or more substituents independently selected fromalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro,hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino,acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and wherein anytwo atoms in the chain may be taken together with the substituents boundthereto to form a ring, wherein the ring may be further substitutedand/or fused to one or more optionally substituted carbocyclic,heterocyclic, aryl or heteroaryl rings; and wherein when the PIS hasformula (A) the presenter protein is a member of the FKBP family, andwhen the PIS has formula (B) or (C), the presenter protein is a memberof the cyclophilin family.
 2. The method of claim 1, wherein thepresenter protein is a member of the FKBP family.
 3. The method of claim2, wherein the PIS has the structure of formula (A).
 4. The method ofclaim 1, wherein the presenter protein is a member of the cyclophilinfamily.
 5. The method of claim 4, wherein the PIS has the structure offormula (B).
 6. The method of claim 4, wherein the PIS has the structureof formula (C).
 7. The method of claim 1, wherein the complex bindsH-Ras and the TIS contacts one or more corresponding interacting siteson H-Ras.
 8. The method of claim 7, wherein the complex binds H-Ras withat least a two-fold increase in affinity relative to the affinity of thecompound for H-Ras in the absence of the presenter protein.
 9. Themethod of claim 1, wherein the complex binds N-Ras and the TIS contactsone or more corresponding interacting sites on N-Ras.
 10. The method ofclaim 9, wherein the complex binds N-Ras with at least a two-foldincrease in affinity relative to the affinity of the compound for N-Rasin the absence of the presenter protein.